Isolation and purification of antibodies using protein a affinity chromatography

ABSTRACT

Disclosed herein are methods for the isolation and purification of antibodies wherein the use of an affinity chromatographic step results in an antibody composition sufficiently pure for pharmaceutical uses. The methods described herein comprise pH viral reduction/inactivation, ultrafiltration/diafiltration, affinity chromatography, preferably Protein A affinity, ion exchange chromatography, and hydrophobic chromatography. Further, the present invention is directed toward pharmaceutical compositions comprising one or more antibodies of the present invention.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Ser.No. 61/196,753, filed Oct. 20, 2008, which is hereby incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

Purification processes for pharmaceutical grade monoclonal antibodiesproduced by fermentation culture typically involve four basic steps.These steps include (1) harvest/clarification—separation of host cellsfrom the fermentation culture; (2) capture—separation of antibody fromthe majority of components in the clarified harvest; (3) finepurification—removal of residual host cell contaminants and aggregates;and (4) formulation—place the antibody into an appropriate carrier formaximum stability and shelf life.

However, these steps often do not necessarily result in antibodycompositions of sufficient purity for use in pharmaceutical contexts.There is a present need for methods of producing and purifying anantibody of interest in sufficiently pure form to be suitable forpharmaceutical use. The present invention addresses this need.

SUMMARY OF THE INVENTION

The present invention is directed to methods for isolating and purifyingantibodies from a sample matrix. In certain aspects, the invention isdirected to methods of antibody purification employing affinitychromatography, preferably Protein A chromatography. In specificaspects, the methods herein employ an acid inactivation step, anaffinity chromatographich step, and one or more additionalchromatography and/or filtration steps. The chromatography steps caninclude one or more step of ion exchange chromatography and/orhydrophobic interaction chromatography. Further, the present inventionis directed toward pharmaceutical compositions comprising one or moreantibodies purified by a method described herein.

One embodiment or the present invention is directed toward a method ofpurifying an antibody or antigen-binding portion thereof from a samplematrix such that the resulting antibody composition is substantiallyfree of host cell proteins (“HCPs”). In one aspect, the sample matrix(or simply “sample”) comprises a cell line harvest wherein the cell lineis employed to produce specific antibodies of the present invention. Ina particular aspect, the sample matrix is prepared from a cell line usedto produce anti-IL-12 antibodies; in another aspect, the sample matrixis prepared from a cell line used to produce anti-TNFα antibodies; andin another aspect the sample matrix is prepared from a cell line used toproudce anti-IL-18 antibodies.

One method of the present invention involves subjecting a sample matrixcomprising the putative antibody of interest or antigen-binding portionthereof to a pH adjustment. In one aspect, the pH is adjusted to anacidic pH. An example of a suitable pH is between about a pH of 3 and 5,preferably a pH of about 3.5. This primary recovery is performed, inpart, to reduce or inactivate pH-sensitive viruses. In addition toreducing and/or inactivating viruses, the acidic conditions facilitatethe removal of cells and cellular debris thus forming a primary recoverysample. After a suitable period of time, the pH can be adjusted toward amore neutral or basic pH and the primary recovery sample is subjected toaffinity chromatography, preferably Protein A chromatography. In oneaspect, the affinity chromatography sample is collected and furthersubjected to subsequent chromatographic steps such as ion exchange andhydrophobic interactive chromatography.

In one embodiment, the affinity chromatography step comprises subjectingthe primary recovery sample to a column comprising a suitable affinitychromatographic support. Non-limiting examples of such chromatographicsupports include, but are not limited to Protein A resin, Protein Gresin, affinity supports comprising the antigen against which theantibody of interest was raised, and affinity supports comprising an Fcbinding protein. Protein A resin is useful for affinity purification andisolation of antibodies (IgG). In one aspect, a Protein A column isequilibrated with a suitable buffer prior to sample loading. An exampleof a suitable buffer is a Tris/NaCl buffer, pH around 7.2. Followingthis equilibration, the sample can be loaded onto the column. Followingthe loading of the column, the column can be washed one or multipletimes using, e.g., the equilibrating buffer. Other washes includingwashes employing different buffers can be used before eluting thecolumn. The Protein A column can then be eluted using an appropriateelution buffer. An example of a suitable elution buffer is an aceticacid/NaCl buffer, pH around 3.5. The eluate can be monitored usingtechniques well known to those skilled in the art. For example, theabsorbance at OD₂₈₀ can be followed. The eluated fraction(s) of interestcan then be prepared for further processing.

In one embodiment, an ion exchange step follows Protein A affinitychromatography. This ion exchange step can be either cation or anionexchange or a combination of both. This step can be a single ionexchange procedure or can include multiple ion exchange steps such as acation exchange step followed by an anion exchange step or visa versa.In one aspect, the ion exchange step is a one step procedure. In anotheraspect, the ion exchange step involves a two step ion exchange process.A suitable cation exchange column is a column whose stationary phasecomprises anionic groups. An example of such a column is a Fractogel™SO₃ ⁻. This ion exchange capture chromatography step facilitates theisolation of antibodies from a sample. A suitable anion exchange columnis a column whose stationary phase comprises cationic groups. An exampleof such a column is a Q Sepharose™ column. One or more ion exchange stepfurther isolates antibodies by reducing impurities such as host cellproteins and DNA and, where applicable, affinity matrix protein. Thisanion exchange procedure is a flow through mode of chromatographywherein the antibodies of interest do not interact or bind to the anionexchange resin (or solid phase). However, many impurities do interactwith and bind to the anion exchange resin. In a particular aspect, theion exchange step is anion exchange chromatography.

The affinity chromatography eluate is prepared for ion exchange byadjusting the pH and ionic strength of the sample buffer. For example,the affinity eluate can be adjusted to a pH of about 6.0 to about 8.5 ina 1 M Tris buffer. Prior to loading the sample (the affinity eluate)onto the ion exchange column, the column can be equilibrated using asuitable buffer. An example of a suitable buffer is a Tris/NaCl bufferwith a pH of about 6.0 to about 8. Following equilibration, the columncan be loaded with the affinity eluate. Following loading, the columncan be washed one or multiple times with a suitable buffer. An exampleof a suitable buffer is the equilibration buffer itself. Flow-throughcollection can commence, e.g., as the absorbance (OD₂₈₀) rises aboveabout 0.2 AU.

In certain embodiments, the present method employs a further step. Thisstep involves the use of hydrophobic interactive chromatography (“HIC”).A suitable column is one whose stationary phase comprises hydrophobicgroups. An example of such a column is a phenyl Sepharose™ column. It ispossible that the antibodies of interest have formed aggregates duringthe isolation/purification process. This hydrophobic chromatographicstep facilitates the elimination of these aggregations. It also assistsin the removal of impurities. The procedure uses a high salt bufferwhich promotes interaction of the antibodies (or aggregations thereof)with the hydrophobic column. The column is eluted using lowerconcentrations of salt.

In another embodiment, the HIC eluate is filtered using a viral removalfilter such as an Ultipor DV20™ filter. This procedure separates viralparticles from the phenyl eluate to reduce the amount of virus (ifpresent) to safe levels. Filters well known to those skilled in the artcan be used in this embodiment.

The purity of the antibodies of interest in the resultant sample productcan be analyzed using methods well known to those skilled in the art,e.g., size-exclusion chromatography, Poros™ A HPLC Assay, HCP ELISA,Protein A ELISA, and western blot analysis.

In yet another embodiment, the invention is directed to one or morepharmaceutical compositions comprising an isolated antibody orantigen-binding portion thereof and an acceptable carrier. In anotheraspect, the compositions further comprise one or more pharmaceuticalagents.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 discloses the heavy and light chain variable region sequences ofa non-limiting example of an anti-IL-12 antibody (ABT-847).

FIG. 2 discloses the heavy and light chain sequences of a non-limitingexample of an anti-IL-18 antibody (ABT-325).

FIG. 3 discloses the heavy and light chain variable region sequences ofa non-limiting example of an anti-TNFα antibody (Adalimumab).

FIG. 4 depicts a representative chromatogram from a 300 L bioreactorshowing product purity after MabSelect™ Protein A chromatography wasused to capture anti-IL-12 antibodies and reduce product and processrelated impurities such as single and double light chain fragments, CHOhost cell proteins (HCPs) and DNA, etc.

FIG. 5 depicts an evaluation of MabSelect™ wash conditions.

FIG. 6 is a photograph of a polyacrylamide gel of precipitates obtainedafter a MabSelect™ eluate was adjusted to pH 8 and a conductivity of 7mS/cm.

FIG. 7 depicts a representative flow-through wash profile of a 300 Lbioreactor scale Q Sepharose™ FF chromatography column.

FIG. 8 depicts a representative elution profile of a 300 L bioreactorscale Phenyl Sepharose™ HP chromatography column.

FIG. 9 depicts the results of a dynamic binding capacity assay where the10% breakthrough point for MabSelect™ resin is identified to be at 37.4g antibody per L of resin.

FIG. 10 is a photograph of a polyacrylamide gel conducted to compare theintermediate differences between Protein A process and AY-04 process. 2μg of antibody of each sample was loaded.

FIG. 11 depicts MabSelect™ Adalimumab Recovery Yield vs. Elutin pH. Thefigure demonstrates that a yield of at least 90% was obtained for anelution pH range from 2.5 to 3.8 and a significant decrease in yieldoccurs at pH 4.

FIG. 12 depicts the results of an assay comparing the recovery ofAdalimumab monomer vs. elution pH and incubation time.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to methods for isolating and purifyingantibodies from a sample matrix. One aspect of the invention is directedto viral reduction/inactivation of samples generated in the varioussteps of antibody purification. In a particular aspect, methods hereinemploy an acid viral reduction/inactivation step followed by one or morechromatography steps. The chromatography steps can include one or moreof the following chromatographic procedures: ion exchangechromatography, affinity chromatography, and hydrophobic interactionchromatography. Further, the present invention is directed towardpharmaceutical compositions comprising one or more antibodies purifiedby a method described herein.

For clarity and not by way of limitation, this detailed description isdivided into the following sub-portions:

1. Definitions;

2. Antibody Generation;

3. Antibody Production;

4. Antibody Purification;

5. Methods of Assaying Sample Purity;

6. Further Modifications;

7. Pharmaceutical Compositions; and

8. Antibody Uses.

1. Definitions

In order that the present invention may be more readily understood,certain terms are first defined.

The term “antibody” includes an immunoglobulin molecule comprised offour polypeptide chains, two heavy (H) chains and two light (L) chainsinter-connected by disulfide bonds. Each heavy chain is comprised of aheavy chain variable region (abbreviated herein as HCVR or VH) and aheavy chain constant region (CH). The heavy chain constant region iscomprised of three domains, CH1, CH2 and CH3. Each light chain iscomprised of a light chain variable region (abbreviated herein as LCVRor VL) and a light chain constant region. The light chain constantregion is comprised of one domain, CL. The VH and VL regions can befurther subdivided into regions of hypervariability, termedcomplementarity determining regions (CDRs), interspersed with regionsthat are more conserved, termed framework regions (FR). Each VH and VLis composed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

The term “antigen-binding portion” of an antibody (or “antibodyportion”) includes fragments of an antibody that retain the ability tospecifically bind to an antigen (e.g., hIL-12, hTNFα, or hIL-18). It hasbeen shown that the antigen-binding function of an antibody can beperformed by fragments of a full-length antibody. Examples of bindingfragments encompassed within the term “antigen-binding portion” of anantibody include (i) a Fab fragment, a monovalent fragment comprisingthe VL, VH, CL and CH1 domains; (ii) a F(ab′)₂ fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment comprising the VH and CH1 domains;(iv) a Fv fragment comprising the VL and VH domains of a single arm ofan antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546,the entire teaching of which is incorporated herein by reference), whichcomprises a VH domain; and (vi) an isolated complementarity determiningregion (CDR). Furthermore, although the two domains of the Fv fragment,VL and VH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see, e.g., Birdet al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85:5879-5883, the entire teachings of which areincorporated herein by reference). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding portion” ofan antibody. Other forms of single chain antibodies, such as diabodiesare also encompassed. Diabodies are bivalent, bispecific antibodies inwhich VH and VL domains are expressed on a single polypeptide chain, butusing a linker that is too short to allow for pairing between the twodomains on the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123, theentire teachings of which are incorporated herein by reference). Stillfurther, an antibody or antigen-binding portion thereof may be part of alarger immunoadhesion molecule, formed by covalent or non-covalentassociation of the antibody or antibody portion with one or more otherproteins or peptides. Examples of such immunoadhesion molecules includeuse of the streptavidin core region to make a tetrameric scFv molecule(Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas6:93-101, the entire teaching of which is incorporated herein byreference) and use of a cysteine residue, a marker peptide and aC-terminal polyhistidine tag to make bivalent and biotinylated scFvmolecules (Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31:1047-1058,the entire teaching of which is incorporated herein by reference).Antibody portions, such as Fab and F(ab′)₂ fragments, can be preparedfrom whole antibodies using conventional techniques, such as papain orpepsin digestion, respectively, of whole antibodies. Moreover,antibodies, antibody portions and immunoadhesion molecules can beobtained using standard recombinant DNA techniques, as described herein.In one aspect, the antigen binding portions are complete domains orpairs of complete domains.

The phrase “human interleukin 12” (abbreviated herein as hIL-12, orIL-12), as used herein, includes a human cytokine that is secretedprimarily by macrophages and dendritic cells. The term includes aheterodimeric protein comprising a 35 kD subunit (p35) and a 40 kDsubunit (p40) which are linked together with a disulfide bridge. Theheterodimeric protein is referred to as a “p70 subunit”. The structureof human IL-12 is described further in, e.g., Kobayashi, et al. (1989)J. Exp Med. 170:827-845; Seder, et al. (1993) Proc. Natl. Acad. Sci.90:10188-10192; Ling, et al. (1995) J. Exp Med. 154:116-127; Podlaski,et al. (1992) Arch. Biochem. Biophys. 294:230-237, the entire teachingsof which are incorporated herein by reference. The nucleic acid encodingIL-12 is available as GenBank Accession No. NM_(—)000882 and thepolypeptide sequence is available as GenBank Accession No.NP_(—)000873.2. The term human IL-12 is intended to include recombinanthuman IL-12 (rh IL-12), which can be prepared by standard recombinantexpression methods.

The phrase “human interleukin 18” (abbreviated herein as hIL-18, orIL-18), as used herein, includes a human cytokine that is initiallysynthesized as biologically inactive 193 amino acid precursor protein aswell as the 156 amino acid mature protein produced by, for example, butnot by way of limitation, cleavage of the precursor protein, e.g., bycaspase-1 or caspase-4, which exhibits biological activities thatinclude the co-stimulation of T cell proliferation, the enhancement ofNK cell cytotoxicity, the induction of IFN-γ production by T cells andNK cells, and the potentiation of T helper type 1 (Th1) differentiation.The nucleic acid encoding IL-18 is available as GenBank Accession No.NM_(—)001562 and the polypeptide sequence is available as GenBankAccession No. NP_(—)001553. The term human IL-18 is intended to includerecombinant human IL-18 (rh IL-18), which can be prepared by standardrecombinant expression methods.

The phrase “human Tumor necrosis factor-α” (abbreviated herein as hTNFαor TNFα) is a multifunctional pro-inflammatory cytokine secretedpredominantly by monocytes/macrophages that has effects on lipidmetabolism, coagulation, insulin resistance, and endothelial function.TNFα is a soluble homotrimer of 17 kD protein subunits. A membrane-bound26 kD precursor form of TNFα also exists. It is found in synovial cellsand macrophages in tissues. Cells other than monocytes or macrophagesalso produce TNFα. For example, human non-monocytic tumor cell linesproduce TNFα as well as CD4+ and CD8+ peripheral blood T lymphocytes andsome cultured T and B cell lines produce TNFα. The nucleic acid encodingTNFα is available as GenBank Accession No. X02910 and the polypeptidesequence is available as GenBank Accession No. CAA26669. The term humanTNFα is intended to include recombinant human TNFα (rh TNFα), which canbe prepared by standard recombinant expression methods.

The terms “Kabat numbering”, “Kabat definitions” and “Kabat labeling”are used interchangeably herein. These terms, which are recognized inthe art, refer to a system of numbering amino acid residues which aremore variable (i.e., hypervariable) than other amino acid residues inthe heavy and light chain variable regions of an antibody, or an antigenbinding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci.190:382-391 and, Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242, the entire teachings ofwhich are incorporated herein by reference). For the heavy chainvariable region, the hypervariable region ranges from amino acidpositions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, andamino acid positions 95 to 102 for CDR3. For the light chain variableregion, the hypervariable region ranges from amino acid positions 24 to34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acidpositions 89 to 97 for CDR3.

The term “human antibody” includes antibodies having variable andconstant regions corresponding to human germline immunoglobulinsequences as described by Kabat et al. (See Kabat, et al. (1991)Sequences of proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242).The human antibodies of the invention may include amino acid residuesnot encoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo), e.g., in the CDRs and in particular CDR3. Themutations can be introduced using the “selective mutagenesis approach.”The human antibody can have at least one position replaced with an aminoacid residue, e.g., an activity enhancing amino acid residue which isnot encoded by the human germline immunoglobulin sequence. The humanantibody can have up to twenty positions replaced with amino acidresidues which are not part of the human germline immunoglobulinsequence. In other embodiments, up to ten, up to five, up to three or upto two positions are replaced. In one embodiment, these replacements arewithin the CDR regions. However, the term “human antibody”, as usedherein, is not intended to include antibodies in which CDR sequencesderived from the germline of another mammalian species, such as a mouse,have been grafted onto human framework sequences.

The phrase “selective mutagenesis approach” includes a method ofimproving the activity of an antibody by selecting and individuallymutating CDR amino acids at least one suitable selective mutagenesisposition, hypermutation, and/or contact position. A “selectivelymutated” human antibody is an antibody which comprises a mutation at aposition selected using a selective mutagenesis approach. In anotheraspect, the selective mutagenesis approach is intended to provide amethod of preferentially mutating selected individual amino acidresidues in the CDR1, CDR2 or CDR3 of the heavy chain variable region(hereinafter H1, H2, and H3, respectively), or the CDR1, CDR2 or CDR3 ofthe light chain variable region (hereinafter referred to as L1, L2, andL3, respectively) of an antibody. Amino acid residues may be selectedfrom selective mutagenesis positions, contact positions, orhypermutation positions. Individual amino acids are selected based ontheir position in the light or heavy chain variable region. It should beunderstood that a hypermutation position can also be a contact position.In one aspect, the selective mutagenesis approach is a “targetedapproach”. The language “targeted approach” is intended to include amethod of mutating selected individual amino acid residues in the CDR1,CDR2 or CDR3 of the heavy chain variable region or the CDR1, CDR2 orCDR3 of the light chain variable region of an antibody in a targetedmanner, e.g., a “Group-wise targeted approach” or “CDR-wise targetedapproach”. In the “Group-wise targeted approach”, individual amino acidresidues in particular groups are targeted for selective mutationsincluding groups I (including L3 and H3), II (including H2 and L1) andIII (including L2 and H1), the groups being listed in order ofpreference for targeting. In the “CDR-wise targeted approach”,individual amino acid residues in particular CDRs are targeted forselective mutations with the order of preference for targeting asfollows: H3, L3, H2, L1, H1 and L2. The selected amino acid residue ismutated, e.g., to at least two other amino acid residues, and the effectof the mutation on the activity of the antibody is determined. Activityis measured as a change in the binding specificity/affinity of theantibody, and/or neutralization potency of the antibody. It should beunderstood that the selective mutagenesis approach can be used for theoptimization of any antibody derived from any source including phagedisplay, transgenic animals with human IgG germline genes, humanantibodies isolated from human B-cells. The selective mutagenesisapproach can be used on antibodies which can not be optimized furtherusing phage display technology. It should be understood that antibodiesfrom any source including phage display, transgenic animals with humanIgG germline genes, human antibodies isolated from human B-cells can besubject to back-mutation prior to or after the selective mutagenesisapproach.

The phrase “recombinant human antibody” includes human antibodies thatare prepared, expressed, created or isolated by recombinant means, suchas antibodies expressed using a recombinant expression vectortransfected into a host cell, antibodies isolated from a recombinant,combinatorial human antibody library, antibodies isolated from an animal(e.g., a mouse) that is transgenic for human immunoglobulin genes (see,e.g., Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295, theentire teaching of which is incorporated herein by reference) orantibodies prepared, expressed, created or isolated by any other meansthat involves splicing of human immunoglobulin gene sequences to otherDNA sequences. Such recombinant human antibodies have variable andconstant regions derived from human germline immunoglobulin sequences(see, Kabat, E. A., et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242). In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the VH and VLregions of the recombinant antibodies are sequences that, while derivedfrom and related to human germline VH and VL sequences, may notnaturally exist within the human antibody germline repertoire in vivo.In certain embodiments, however, such recombinant antibodies are theresult of selective mutagenesis approach or back-mutation or both.

An “isolated antibody” includes an antibody that is substantially freeof other antibodies having different antigenic specificities (e.g., anisolated antibody that specifically binds hIL-12 is substantially freeof antibodies that specifically bind antigens other than hIL-12). Anisolated antibody that specifically binds hIL-12 may bind IL-12molecules from other species. Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals. Suitableanti-IL-12 antibodies that may be purified in the context of the instantinvention are disclosed in U.S. Pat. No. 6,914,128 (which is herebyincorporated by reference in its entirety) including, but not limited tothe anti-IL-12 antibody identified in that patent as J695, and which hassubsequently been identified as ABT-874. Suitable anti-IL-18 antibodiesthat may be purified and isolated in the context of the instantinvention are disclosed in U.S. Ser. Nos. 09/780,035 and 10/988,360,including, the antibody that has subsequently been identified asABT-325. A suitible anti-TNFα antibody is Adalimumab (AbbottLaboratories).

A “neutralizing antibody” (or an “antibody that neutralized hIL-12activity”) includes an antibody whose binding to hIL-12 results ininhibition of the biological activity of hIL-12. This inhibition of thebiological activity of hIL-12 can be assessed by measuring one or moreindicators of hIL-12 biological activity, such as inhibition of humanphytohemagglutinin blast proliferation in a phytohemagglutinin blastproliferation assay (PHA), or inhibition of receptor binding in a humanIL-12 receptor binding assay. These indicators of hIL-12 biologicalactivity can be assessed by one or more of several standard in vitro orin vivo assays known in the art.

A “neutralizing antibody” (or an “antibody that neutralized hIL-18activity”) includes an antibody whose binding to hIL-18 results ininhibition of the biological activity of hIL-18. This inhibition of thebiological activity of hIL-18 can be assessed by measuring one or moreindicators of hIL-18 biological activity, such as induction of IFNγproduction by T cells or NK cells, or inhibition of IL-18 receptorbinding in a human IL-18 receptor binding assay. These indicators ofhIL-18 biological activity can be assessed by one or more of severalstandard in vitro or in vivo assays known in the art.

The term “activity” includes activities such as the bindingspecificity/affinity of an antibody for an antigen, e.g., an anti-hIL-12antibody that binds to an IL-12 antigen and/or the neutralizing potencyof an antibody, e.g., an anti-hIL-12 antibody whose binding to hIL-12inhibits the biological activity of hIL-12, e.g., inhibition of PHAblast proliferation or inhibition of receptor binding in a human IL-12receptor binding assay. The term “activity” also includes activitiessuch as the binding specificity/affinity of an anti-IL-18 antibody forits antigen, e.g., an anti-hIL-18 antibody that binds to an IL-18antigen and/or the neutralizing potency of an antibody, e.g., ananti-hIL-18 antibody whose binding to hIL-18 inhibits the biologicalactivity of hIL-18. The term “activity” also includes activities such asthe binding specificity/affinity of an anti-TNFα antibody for itsantigen, e.g., an anti-TNFα antibody that binds to a TNFα antigen and/orthe neutralizing potency of an antibody, e.g., an anti-TNFα antibodywhose binding to hTNFα inhibits the biological activity of hTNFα.

The phrase “surface plasmon resonance” includes an optical phenomenonthat allows for the analysis of real-time biospecific interactions bydetection of alterations in protein concentrations within a biosensormatrix, e.g., using the BIAcore system (Pharmacia Biosensor AB, Uppsala,Sweden and Piscataway, N.J.). For further descriptions, see Jonsson, U.,et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., et al. (1991)Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit.8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277,the entire teachings of which are incorporated herein.

The term “Koff”, as used herein, is intended to refer to the off rateconstant for dissociation of an antibody from the antibody/antigencomplex.

The term “Kd”, as used herein, is intended to refer to the dissociationconstant of a particular antibody-antigen interaction.

The phrase “nucleic acid molecule” includes DNA molecules and RNAmolecules. A nucleic acid molecule may be single-stranded ordouble-stranded, but in one aspect is double-stranded DNA.

The phrase “isolated nucleic acid molecule,” as used herein in referenceto nucleic acids encoding antibodies or antibody portions (e.g., VH, VL,CDR3), e.g. those that bind hIL-12, hTNFα, or hIL-18, and includes anucleic acid molecule in which the nucleotide sequences encoding theantibody or antibody portion are free of other nucleotide sequencesencoding antibodies or antibody portions that bind antigens other thanhIL-12, hTNFα, or hIL-18, which other sequences may naturally flank thenucleic acid in human genomic DNA. Thus, e.g, an isolated nucleic acidof the invention encoding a VH region of an anti-IL-12 h, anti-TNFα, oranti-hIL-18 antibody contains no other sequences encoding other VHregions that bind antigens other than, for example, IL-12, hTNFα, orhIL-18. The phrase “isolated nucleic acid molecule” is also intended toinclude sequences encoding bivalent, bispecific antibodies, such asdiabodies in which VH and VL regions contain no other sequences otherthan the sequences of the diabody.

The phrase “recombinant host cell” (or simply “host cell”) includes acell into which a recombinant expression vector has been introduced. Itshould be understood that such terms are intended to refer not only tothe particular subject cell but to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein.

The term “modifying”, as used herein, is intended to refer to changingone or more amino acids in the antibodies or antigen-binding portionsthereof. The change can be produced by adding, substituting or deletingan amino acid at one or more positions. The change can be produced usingknown techniques, such as PCR mutagenesis.

The term “about”, as used herein, is intended to refer to ranges ofapproximately 10-20% greater than or less than the referenced value. Incertain circumstances, one of skill in the art will recognize that, dueto the nature of the referenced value, the term “about” can mean more orless than a 10-20% deviation from that value.

The phrase “viral reduction/inactivation”, as used herein, is intendedto refer to a decrease in the number of viral particles in a particularsample (“reduction”), as well as a decrease in the activity, forexample, but not limited to, the infectivity or ability to replicate, ofviral particles in a particular sample (“inactivation”). Such decreasesin the number and/or activity of viral particles can be on the order ofabout 1% to about 99%, preferably of about 20% to about 99%, morepreferably of about 30% to about 99%, more preferably of about 40% toabout 99%, even more preferably of about 50% to about 99%, even morepreferably of about 60% to about 99%, yet more preferably of about 70%to about 99%, yet more preferably of about 80% to 99%, and yet morepreferably of about 90% to about 99%. In certain non-limitingembodiments, the amount of virus, if any, in the purified antibodyproduct is less than the ID50 (the amount of virus that will infect 50percent of a target population) for that virus, preferably at least10-fold less than the ID50 for that virus, more preferably at least100-fold less than the ID50 for that virus, and still more preferably atleast 1000-fold less than the ID50 for that virus

The phrase “contact position” includes an amino acid position in theCDR1, CDR2 or CDR3 of the heavy chain variable region or the light chainvariable region of an antibody which is occupied by an amino acid thatcontacts antigen in one of the twenty-six known antibody-antigenstructures. If a CDR amino acid in any of the twenty-six known solvedstructures of antibody-antigen complexes contacts the antigen, then thatamino acid can be considered to occupy a contact position. Contactpositions have a higher probability of being occupied by an amino acidwhich contact antigens than in a non-contact position. In one aspect, acontact position is a CDR position which contains an amino acid thatcontacts antigen in greater than 3 of the 26 structures (>1.5%). Inanother aspect, a contact position is a CDR position which contains anamino acid that contacts antigen in greater than 8 of the 25 structures(>32%).

2. Antibody Generation

The term “antibody” as used in this section refers to an intact antibodyor an antigen binding fragment thereof.

The antibodies of the present disclosure can be generated by a varietyof techniques, including immunization of an animal with the antigen ofinterest followed by conventional monoclonal antibody methodologiese.g., the standard somatic cell hybridization technique of Kohler andMilstein (1975) Nature 256: 495. Although somatic cell hybridizationprocedures are preferred, in principle, other techniques for producingmonoclonal antibody can be employed e.g., viral or oncogenictransformation of B lymphocytes.

One preferred animal system for preparing hybridomas is the murinesystem. Hybridoma production is a very well-established procedure.Immunization protocols and techniques for isolation of immunizedsplenocytes for fusion are known in the art. Fusion partners (e.g.,murine myeloma cells) and fusion procedures are also known.

An antibody preferably can be a human, a chimeric, or a humanizedantibody. Chimeric or humanized antibodies of the present disclosure canbe prepared based on the sequence of a non-human monoclonal antibodyprepared as described above. DNA encoding the heavy and light chainimmunoglobulins can be obtained from the non-human hybridoma of interestand engineered to contain non-murine (e.g., human) immunoglobulinsequences using standard molecular biology techniques. For example, tocreate a chimeric antibody, murine variable regions can be linked tohuman constant regions using methods known in the art (see e.g., U.S.Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody,murine CDR regions can be inserted into a human framework using methodsknown in the art (see e.g., U.S. Pat. No. 5,225,539 to Winter, and U.S.Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen etal.).

In one non-limiting embodiment, the antibodies of this disclosure arehuman monoclonal antibodies. Such human monoclonal antibodies directedagainst IL-12, TNFα, or IL-18 can be generated using transgenic ortranschromosomic mice carrying parts of the human immune system ratherthan the mouse system. These transgenic and transchromosomic miceinclude mice referred to herein as the HuMAb Mouse® (Medarex, Inc.), KMMouse® (Medarex, Inc.), and XenoMouse® (Amgen).

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseantibodies of the disclosure, such as anti-IL-12, anti-TNFα, oranti-IL-18 antibodies. For example, mice carrying both a human heavychain transchromosome and a human light chain tranchromosome, referredto as “TC mice” can be used; such mice are described in Tomizuka et al.(2000) Proc. Natl. Acad. Sci. USA 97:722-727. Furthermore, cows carryinghuman heavy and light chain transchromosomes have been described in theart (e.g., Kuroiwa et al. (2002) Nature Biotechnology 20:889-894 and PCTapplication No. WO 2002/092812) and can be used to raise anti-IL-12,anti-TNFα, or anti-IL18 antibodies of this disclosure.

Recombinant human antibodies of the invention, including, but notlimited to, anti-IL-12, anti-TNFα, or anti-IL-18 antibodies or anantigen binding portion thereof, or anti-IL-12-related,anti-TNFα-related, or anti-IL-18-related antibodies disclosed herein canbe isolated by screening of a recombinant combinatorial antibodylibrary, e.g., a scFv phage display library, prepared using human VL andVH cDNAs prepared from mRNA derived from human lymphocytes.Methodologies for preparing and screening such libraries are known inthe art. In addition to commercially available kits for generating phagedisplay libraries (e.g., the Pharmacia Recombinant Phage AntibodySystem, catalog no. 27-9400-01; and the Stratagene SurfZAP™ phagedisplay kit, catalog no. 240612, the entire teachings of which areincorporated herein), examples of methods and reagents particularlyamenable for use in generating and screening antibody display librariescan be found in, e.g., Ladner et al. U.S. Pat. No. 5,223,409; Kang etal. PCT Publication No. WO 92/18619; Dower et al. PCT Publication No. WO91/17271; Winter et al. PCT Publication No. WO 92/20791; Markland et al.PCT Publication No. WO 92/15679; Breitling et al. PCT Publication No. WO93/01288; McCafferty et al. PCT Publication No. WO 92/01047; Garrard etal. PCT Publication No. WO 92/09690; Fuchs et al. (1991) Bio/Technology9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse etal. (1989) Science 246:1275-1281; McCafferty et al., Nature (1990)348:552-554; Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins et al.(1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrard et al. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982; the entireteachings of which are incorporated herein.

Human monoclonal antibodies of this disclosure can also be preparedusing SCID mice into which human immune cells have been reconstitutedsuch that a human antibody response can be generated upon immunization.Such mice are described in, for example, U.S. Pat. Nos. 5,476,996 and5,698,767 to Wilson et al.

In certain embodiments, the methods of the invention include anti-IL-12,anti-TNFα, or anti-IL-18 antibodies and antibody portions,anti-IL-12-related, anti-TNFα-related, or anti-IL-18-related antibodiesand antibody portions, and human antibodies and antibody portions withequivalent properties to anti-IL-12, anti-TNFα, or anti-IL-18antibodies, such as high affinity binding to hIL-12, hTNFα, or hIL-18with low dissociation kinetics and high neutralizing capacity. In oneaspect, the invention provides treatment with an isolated humanantibody, or an antigen-binding portion thereof, that dissociates fromhIL-12, hTNFα, or hIL-18 with a Kd of about 1×10-8 M or less and a Koffrate constant of 1×10-3 s-1 or less, both determined by surface plasmonresonance. In specific non-limiting embodiments, an anti-IL12 antibodypurified according to the invention competitively inhibits binding ofABT-874 to IL12 under physiological conditions. In specific non-limitingembodiments, an anti-IL-18 antibody purified according to the inventioncompetitively inhibits binding of ABT-325 to IL-18 under physiologicalconditions. In specific non-limiting embodiments, an anti-TNFα antibodypurified according to the invention competitively inhibits binding ofAdalimumab to TNFα under physiological conditions.

In yet another embodiment of the invention, antibodies or fragmentsthereof, such as but not limited to anti-IL-12, anti-TNFα, or anti-IL-18antibodies or fragments thereof, can be altered wherein the constantregion of the antibody is modified to reduce at least one constantregion-mediated biological effector function relative to an unmodifiedantibody. To modify an antibody of the invention such that it exhibitsreduced binding to the Fc receptor, the immunoglobulin constant regionsegment of the antibody can be mutated at particular regions necessaryfor Fc receptor (FcR) interactions (see, e.g., Canfield and Morrison(1991) J. Exp. Med. 173:1483-1491; and Lund et al. (1991) J of Immunol.147:2657-2662, the entire teachings of which are incorporated herein).Reduction in FcR binding ability of the antibody may also reduce othereffector functions which rely on FcR interactions, such as opsonizationand phagocytosis and antigen-dependent cellular cytotoxicity.

3. Antibody Production

To express an antibody of the invention, DNAs encoding partial orfull-length light and heavy chains are inserted into one or moreexpression vector such that the genes are operatively linked totranscriptional and translational control sequences. (See, e.g., U.S.Pat. No. 6,914,128, the entire teaching of which is incorporated hereinby reference.) In this context, the term “operatively linked” isintended to mean that an antibody gene is ligated into a vector suchthat transcriptional and translational control sequences within thevector serve their intended function of regulating the transcription andtranslation of the antibody gene. The expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. The antibody light chain gene and the antibody heavy chaingene can be inserted into a separate vector or, more typically, bothgenes are inserted into the same expression vector. The antibody genesare inserted into an expression vector by standard methods (e.g.,ligation of complementary restriction sites on the antibody genefragment and vector, or blunt end ligation if no restriction sites arepresent). Prior to insertion of the antibody or antibody-related lightor heavy chain sequences, the expression vector may already carryantibody constant region sequences. For example, one approach toconverting the anti-IL-12, anti-TNFα, or anti-IL-18 antibody oranti-IL-12, anti-TNFα, or anti-IL-18 antibody-related VH and VLsequences to full-length antibody genes is to insert them intoexpression vectors already encoding heavy chain constant and light chainconstant regions, respectively, such that the VH segment is operativelylinked to the CH segment(s) within the vector and the VL segment isoperatively linked to the CL segment within the vector. Additionally oralternatively, the recombinant expression vector can encode a signalpeptide that facilitates secretion of the antibody chain from a hostcell. The antibody chain gene can be cloned into the vector such thatthe signal peptide is linked in-frame to the amino terminus of theantibody chain gene. The signal peptide can be an immunoglobulin signalpeptide or a heterologous signal peptide (i.e., a signal peptide from anon-immunoglobulin protein).

In addition to the antibody chain genes, a recombinant expression vectorof the invention can carry one or more regulatory sequence that controlsthe expression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, e.g., in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990), the entire teaching of which is incorporatedherein by reference. It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Suitable regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., theadenovirus major late promoter (AdMLP)) and polyoma. For furtherdescription of viral regulatory elements, and sequences thereof, see,e.g., U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 byBell et al. and U.S. Pat. No. 4,968,615 by Schaffner et al., the entireteachings of which are incorporated herein by reference.

In addition to the antibody chain genes and regulatory sequences, arecombinant expression vector of the invention may carry one or moreadditional sequences, such as a sequence that regulates replication ofthe vector in host cells (e.g., origins of replication) and/or aselectable marker gene. The selectable marker gene facilitates selectionof host cells into which the vector has been introduced (see e.g., U.S.Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al., theentire teachings of which are incorporated herein by reference). Forexample, typically the selectable marker gene confers resistance todrugs, such as G418, hygromycin or methotrexate, on a host cell intowhich the vector has been introduced. Suitable selectable marker genesinclude the dihydrofolate reductase (DHFR) gene (for use in dhfr- hostcells with methotrexate selection/amplification) and the neo gene (forG418 selection).

An antibody, or antibody portion, of the invention can be prepared byrecombinant expression of immunoglobulin light and heavy chain genes ina host cell. To express an antibody recombinantly, a host cell istransfected with one or more recombinant expression vectors carrying DNAfragments encoding the immunoglobulin light and heavy chains of theantibody such that the light and heavy chains are expressed in the hostcell and secreted into the medium in which the host cells are cultured,from which medium the antibodies can be recovered. Standard recombinantDNA methodologies are used to obtain antibody heavy and light chaingenes, incorporate these genes into recombinant expression vectors andintroduce the vectors into host cells, such as those described inSambrook, Fritsch and Maniatis (eds), Molecular Cloning; A LaboratoryManual, Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel et al.(eds.) Current Protocols in Molecular Biology, Greene PublishingAssociates, (1989) and in U.S. Pat. Nos. 4,816,397 & 6,914,128, theentire teachings of which are incorporated herein.

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is (are) transfected into a hostcell by standard techniques. The various forms of the term“transfection” are intended to encompass a wide variety of techniquescommonly used for the introduction of exogenous DNA into a prokaryoticor eukaryotic host cell, e.g., electroporation, calcium-phosphateprecipitation, DEAE-dextran transfection and the like. Although it istheoretically possible to express the antibodies of the invention ineither prokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, such as mammalian host cells, is suitable because sucheukaryotic cells, and in particular mammalian cells, are more likelythan prokaryotic cells to assemble and secrete a properly folded andimmunologically active antibody. Prokaryotic expression of antibodygenes has been reported to be ineffective for production of high yieldsof active antibody (Boss and Wood (1985) Immunology Today 6:12-13, theentire teaching of which is incorporated herein by reference).

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, e.g., Enterobacteriaceae suchas Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella,Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g.,Serratia marcescans, and Shigella, as well as Bacilli such as B.subtilis and B. licheniformis (e.g., B. licheniformis 41P disclosed inDD 266,710 published Apr. 12, 1989), Pseudomonas such as P. aeruginosa,and Streptomyces. One suitable E. coli cloning host is E. coli 294 (ATCC31,446), although other strains such as E. coli B, E. coli X1776 (ATCC31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examplesare illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for polypeptideencoding vectors. Saccharomyces cerevisiae, or common baker's yeast, isthe most commonly used among lower eukaryotic host microorganisms.However, a number of other genera, species, and strains are commonlyavailable and useful herein, such as Schizosaccharomyces pombe;Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424),K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii(ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K.marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida;Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces suchas Schwanniomyces occidentalis; and filamentous fungi such as, e.g.,Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A.nidulans and A. niger.

Suitable host cells for the expression of glycosylated antibodies arederived from multicellular organisms. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyxmori have been identified. A variety of viral strains for transfectionare publicly available, e.g., the L-1 variant of Autographa californicaNPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be usedas the virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells. Plant cell cultures ofcotton, corn, potato, soybean, petunia, tomato, and tobacco can also beutilized as hosts.

Suitable mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr- CHO cells, described in Urlaub and Chasin, (1980) PNAS USA77:4216-4220, used with a DHFR selectable marker, e.g., as described inKaufman and Sharp (1982) Mol. Biol. 159:601-621, the entire teachings ofwhich are incorporated herein by reference), NSO myeloma cells, COScells and SP2 cells. When recombinant expression vectors encodingantibody genes are introduced into mammalian host cells, the antibodiesare produced by culturing the host cells for a period of time sufficientto allow for expression of the antibody in the host cells or secretionof the antibody into the culture medium in which the host cells aregrown. Other examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/−DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2), the entire teachings of which are incorporated herein byreference.

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

The host cells used to produce an antibody may be cultured in a varietyof media. Commercially available media such as Ham's F10™ (Sigma),Minimal Essential Medium™ ((MEM), (Sigma), RPMI-1640 (Sigma), andDulbecco's Modified Eagle's Medium™ ((DMEM), Sigma) are suitable forculturing the host cells. In addition, any of the media described in Hamet al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255(1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. No. Re. 30,985 may beused as culture media for the host cells, the entire teachings of whichare incorporated herein by reference. Any of these media may besupplemented as necessary with hormones and/or other growth factors(such as insulin, transferrin, or epidermal growth factor), salts (suchas sodium chloride, calcium, magnesium, and phosphate), buffers (such asHEPES), nucleotides (such as adenosine and thymidine), antibiotics (suchas gentamycin drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

Host cells can also be used to produce portions of intact antibodies,such as Fab fragments or scFv molecules. It is understood thatvariations on the above procedure are within the scope of the presentinvention. For example, in certain embodiments it may be desirable totransfect a host cell with DNA encoding either the light chain or theheavy chain (but not both) of an antibody of this invention. RecombinantDNA technology may also be used to remove some or all of the DNAencoding either or both of the light and heavy chains that is notnecessary for binding to IL-12, specifically hIL-12, in the context ofanti-IL-12 antibodies or that DNA not necessary for binding to IL-18,specifically hIL-18, in the context of anti-IL-18 antibodies, or thatDNA not necessary for binding to TNFα, specifically hTNFα, in thecontext of anti-TNFα antibodies. The molecules expressed from suchtruncated DNA molecules are also encompassed by the antibodies of theinvention. In addition, bifunctional antibodies may be produced in whichone heavy and one light chain are an antibody of the invention and theother heavy and light chain are specific for an antigen other thanIL-12, TNFα, or IL-18, depending on the specificity of the antibody ofthe invention, by crosslinking an antibody of the invention to a secondantibody by standard chemical crosslinking methods.

In a suitable system for recombinant expression of an antibody, orantigen-binding portion thereof, of the invention, a recombinantexpression vector encoding both the antibody heavy chain and theantibody light chain is introduced into dhfr-CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to CMV enhancer/AdMLP promoter regulatory elements to drive highlevels of transcription of the genes. The recombinant expression vectoralso carries a DHFR gene, which allows for selection of CHO cells thathave been transfected with the vector using methotrexateselection/amplification. The selected transformant host cells arecultured to allow for expression of the antibody heavy and light chainsand intact antibody is recovered from the culture medium. Standardmolecular biology techniques are used to prepare the recombinantexpression vector, transfect the host cells, select for transformants,culture the host cells and recover the antibody from the culture medium.

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. In one aspect, if the antibody is produced intracellularly, as afirst step, the particulate debris, either host cells or lysed cells(e.g., resulting from homogenization), can be removed, e.g., bycentrifugation or ultrafiltration. Where the antibody is secreted intothe medium, supernatants from such expression systems can be firstconcentrated using a commercially available protein concentrationfilter, e.g., an Amicon™ or Millipore Pellicon™ ultrafiltration unit.

Prior to the process of the invention, procedures for purification ofantibodies from cell debris initially depend on the site of expressionof the antibody. Some antibodies can be secreted directly from the cellinto the surrounding growth media; others are made intracellularly. Forthe latter antibodies, the first step of a purification processtypically involves: lysis of the cell, which can be done by a variety ofmethods, including mechanical shear, osmotic shock, or enzymatictreatments. Such disruption releases the entire contents of the cellinto the homogenate, and in addition produces subcellular fragments thatare difficult to remove due to their small size. These are generallyremoved by differential centrifugation or by filtration. Where theantibody is secreted, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, e.g., an Amicon™ or Millipore Pellicon™ultrafiltration unit. Where the antibody is secreted into the medium,the recombinant host cells can also be separated from the cell culturemedium, e.g., by tangential flow filtration. Antibodies can be furtherrecovered from the culture medium using the antibody purificationmethods of the invention.

4. Antibody Purification

4.1 Antibody Purification Generally

The invention provides a method for producing a purified (or“HCP-reduced”) antibody preparation from a mixture comprising anantibody and at least one HCP. The purification process of the inventionbegins at the separation step when the antibody has been produced usingmethods described above and conventional methods in the art. Table 1summarizes one embodiment of a purification scheme. Variations of thisscheme, including, but not limited to, variations where the Protein Aaffinity chromatography step is omitted or the order of the ion exchangesteps is reversed, are envisaged and are within the scope of thisinvention.

TABLE 1 Purification steps with their associated purpose Purificationstep Purpose Primary recovery clarification of sample matrix Affinitychromatography antibody capture, host cell protein and associatedimpurity reduction Cation exchange antibody capture, host cell proteinand chromatography associated impurity reduction ultrafiltration/concentration and buffer exchange diafiltration Anion exchange reductionof host cell proteins and DNA chromatography Phenyl Sepharose ™ HPreduction of antibody aggregates and host chromatography cell proteinsViral filtration removal of large viruses, if present Finalultrafiltration/ concentrate and formulate antibody diafiltration

Once a clarified solution or mixture comprising the antibody has beenobtained, separation of the antibody from the other proteins produced bythe cell, such as HCPs, is performed using a combination of differentpurification techniques, including ion exchange separation step(s) andhydrophobic interaction separation step(s). The separation stepsseparate mixtures of proteins on the basis of their charge, degree ofhydrophobicity, or size. In one aspect of the invention, separation isperformed using chromatography, including cationic, anionic, andhydrophobic interaction. Several different chromatography resins areavailable for each of these techniques, allowing accurate tailoring ofthe purification scheme to the particular protein involved. The essenceof each of the separation methods is that proteins can be caused eitherto traverse at different rates down a column, achieving a physicalseparation that increases as they pass further down the column, or toadhere selectively to the separation medium, being then differentiallyeluted by different solvents. In some cases, the antibody is separatedfrom impurities when the impurities specifically adhere to the columnand the antibody does not, i.e., the antibody is present in the flowthrough.

As noted above, accurate tailoring of a purification scheme relies onconsideration of the protein to be purified. In certain embodiments, theseparation steps of the instant invention are employed to separate anantibody from one or more HCPs. Antibodies that can be successfullypurified using the methods described herein include, but are not limitedto, human IgA₁, IgA₂, IgD, IgE, IgG₁, IgG₂, IgG₃, IgG₄, and IgMantibodies. In certain embodiments, the purification strategies of theinstant invention exclude the use of Protein A affinity chromatography,for example in the context of the purification of IgG₃ antibodies, asIgG₃ antibodies bind to Protein A inefficiently. Other factors thatallow for specific tailoring of a purification scheme include, but arenot limited to: the presence or absence of an Fc region (e.g., in thecontext of full length antibody as compared to an Fab fragment thereof)because Protein A binds to the Fc region; the particular germlinesequences employed in generating to antibody of interest; and the aminoacid composition of the antibody (e.g., the primary sequence of theantibody as well as the overall charge/hydrophobicity of the molecule).Antibodies sharing one or more characteristic can be purified usingpurification strategies tailored to take advantage of thatcharacteristic.

4.2 Primary Recovery

The initial steps of the purification methods of the present inventioninvolve the first phase of clarification and primary recovery ofantibody from a sample matrix. In addition, the primary recovery processcan also be a point at which to reduce or inactivate viruses that can bepresent in the sample matrix. For example, any one or more of a varietyof methods of viral reduction/inactivation can be used during theprimary recovery phase of purification including heat inactivation(pasteurization), pH inactivation, solvent/detergent treatment, UV andγ-ray irradiation and the addition of certain chemical inactivatingagents such as β-propiolactoneor e.g., copper phenanthroline as in U.S.Pat. No. 4,534,972, the entire teaching of which is incorporated hereinby reference. In certain embodiments of the present invention, thesample matrix is exposed to pH viral reduction/inactivation during theprimary recovery phase.

Methods of pH viral reduction/inactivation include, but are not limitedto, incubating the mixture for a period of time at low pH, andsubsequently neutralizing the pH and removing particulates byfiltration. In certain embodiments the mixture will be incubated at a pHof between about 2 and 5, preferably at a pH of between about 3 and 4,and more preferably at a pH of about 3.5. The pH of the sample mixturemay be lowered by any suitable acid including, but not limited to,citric acid, acetic acid, caprylic acid, or other suitable acids. Thechoice of pH level largely depends on the stability profile of theantibody product and buffer components. It is known that the quality ofthe target antibody during low pH virus reduction/inactivation isaffected by pH and the duration of the low pH incubation. In certainembodiments the duration of the low pH incubation will be from 0.5 hr totwo 2 hr, preferably 0.5 hr to 1.5 hr, and more preferably the durationwill be 1 hr. Virus reduction/inactivation is dependent on these sameparameters in addition to protein concentration, which may limitreduction/inactivation at high concentrations. Thus, the properparameters of protein concentration, pH, and duration ofreduction/inactivation can be selected to achieve the desired level ofviral reduction/inactivation.

In certain embodiments viral reduction/inactivation can be achieved viathe use of suitable filters. A non-limiting example of a suitable filteris the Ultipor DV50™ filter from Pall Corporation. Although certainembodiments of the present invention employ such filtration during theprimary recovery phase, in other embodiments it is employed at otherphases of the purification process, including as either the penultimateor final step of purification. In certain embodiments, alternativefilters are employed for viral reduction/inactivation, such as, but notlimited to, Viresolve™ filters (Millipore, Billerica, Mass.); Zeta PlusVR™ filters (CUNO; Meriden, Conn.); and Planova™ filters (Asahi KaseiPharma, Planova Division, Buffalo Grove, Ill.).

In those embodiments where viral reduction/inactivation is employed, thesample mixture can be adjusted, as needed, for further purificationsteps. For example, following low pH viral reduction/inactivation the pHof the sample mixture is typically adjusted to a more neutral pH, e.g.,from about 4.5 to about 8.5, and preferably about 4.9, prior tocontinuing the purification process. Additionally, the mixture may beflushed with water for injection (WFI) to obtain a desired conductivity.

In certain embodiments, the primary recovery will include one or morecentrifugation steps to further clarify the sample matrix and therebyaid in purifying the anti-IL-12, anti-TNFα, or anti-IL-18 antibodies.Centrifugation of the sample can be run at, for example, but not by wayof limitation, 7,000×g to approximately 12,750×g. In the context oflarge scale purification, such centrifugation can occur on-line with aflow rate set to achieve, for example, but not by way of limitation, aturbidity level of 150 NTU in the resulting supernatant. Suchsupernatant can then be collected for further purification.

In certain embodiments, the primary recovery will include the use of oneor more depth filtration steps to further clarify the sample matrix andthereby aid in purifying the antibodies of the present invention. Depthfilters contain filtration media having a graded density. Such gradeddensity allows larger particles to be trapped near the surface of thefilter while smaller particles penetrate the larger open areas at thesurface of the filter, only to be trapped in the smaller openings nearerto the center of the filter. In certain embodiments the depth filtrationstep can be a delipid depth filtration step. Although certainembodiments employ depth filtration steps only during the primaryrecovery phase, other embodiments employ depth filters, includingdelipid depth filters, during one or more additional phases ofpurification. Non-limiting examples of depth filters that can be used inthe context of the instant invention include the Cuno™ model 30/60ZAdepth filters (3M Corp.), and 0.45/0.2 μm Sartopore™ bi-layer filtercartridges.

4.3 Affinity Chromatography

In certain embodiments, the primary recovery sample is subjected toaffinity chromatography to further purify the antibody of interest awayfrom HCPs. In certain embodiments the chromatographic material iscapable of selectively or specifically binding to the antibody ofinterest. Non-limiting examples of such chromatographic materialinclude: Protein A, Protein G, chromatographic material comprosing theantigen bound by the antibody of interest, and chromatographic materialcomprising an Fc binding protein. In specific embodiments, the affinitychromatography step involves subjecting the primary recovery sample to acolumn comprising a suitable Protein A resin. Protein A resin is usefulfor affinity purification and isolation of a variety antibody isotypes,particularly IgG₁, IgG₂, and IgG₄. Protein A is a bacterial cell wallprotein that binds to mammalian IgGs primarily through their Fc regions.In its native state, Protein A has five IgG binding domains as well asother domains of unknown function.

There are several commercial sources for Protein A resin. One suitableresin is MabSelect™ from GE Healthcare. A non-limiting example of asuitable column packed with MabSelect™ is an about 1.0 cm diameter×about21.6 cm long column (˜17 mL bed volume). This size column can be usedfor small scale purifications and can be compared with other columnsused for scale ups. For example, a 20 cm×21 cm column whose bed volumeis about 6.6 L can be used for larger purifications. Regardless of thecolumn, the column can be packed using a suitable resin such asMabSelect™.

In certain embodiments it will be advantageous to identify the dynamicbinding capacity (DBC) of the Protein A resin in order to tailor thepurification to the particular antibody of interest. For example, butnot by way of limitation, the DBC of a MabSelect™ column can bedetermined either by a single flow rate load or dual-flow load strategy.The single flow rate load can be evaluated at a velocity of about 300cm/hr throughout the entire loading period. The dual-flow rate loadstrategy can be determined by loading the column up to about 35 mgprotein/mL resin at a linear velocity of about 300 cm/hr, then reducingthe linear velocity by half to allow longer residence time for the lastportion of the load.

In certain embodiments, the Protein A column can be equilibrated with asuitable buffer prior to sample loading. A non-limiting example of asuitable buffer is a Tris/NaCl buffer, pH of about 7.2. A non-limitingexample of suitable equilibration conditions is 25 mM Tris, 100 mM NaCl,pH of about 7.2. Following this equilibration, the sample can be loadedonto the column. Following the loading of the column, the column can bewashed one or multiple times using, e.g., the equilibrating buffer.Other washes, including washes employing different buffers, can beemployed prior to eluting the column. For example, the column can bewashed using one or more column volumes of 20 mM citric acid/sodiumcitrate, 0.5 M NaCl at pH of about 6.0. This wash can optionally befollowed by one or more washes using the equilibrating buffer. TheProtein A column can then be eluted using an appropriate elution buffer.A non-limiting example of a suitable elution buffer is an aceticacid/NaCl buffer, pH of about 3.5. Suitable conditions are, e.g., 0.1 Macetic acid, pH of about 3.5. The eluate can be monitored usingtechniques well known to those skilled in the art. For example, theabsorbance at OD₂₈₀ can be followed. Column eluate can be collectedstarting with an initial deflection of about 0.5 AU to a reading ofabout 0.5 AU at the trailing edge of the elution peak. The elutionfraction(s) of interest can then be prepared for further processing. Forexample, the collected sample can be titrated to a pH of about 5.0 usingTris (e.g., 1.0 M) at a pH of about 10. Optionally, this titrated samplecan be filtered and further processed.

4.4 Ion Exchange Chromatography

In certain embodiments, the instant invention provides methods forproducing a HCP-reduced antibody preparation from a mixture comprisingan antibody and at least one HCP by subjecting the mixture to at leastone ion exchange separation step such that an eluate comprising theantibody is obtained. Ion exchange separation includes any method bywhich two substances are separated based on the difference in theirrespective ionic charges, and can employ either cationic exchangematerial or anionic exchange material.

The use of a cationic exchange material versus an anionic exchangematerial is based on the overall charge of the protein. Therefore, it iswithin the scope of this invention to employ an anionic exchange stepprior to the use of a cationic exchange step, or a cationic exchangestep prior to the use of an anionic exchange step. Furthermore, it iswithin the scope of this invention to employ only a cationic exchangestep, only an anionic exchange step, or any serial combination of thetwo.

In performing the separation, the initial antibody mixture can becontacted with the ion exchange material by using any of a variety oftechniques, e.g., using a batch purification technique or achromatographic technique.

For example, in the context of batch purification, ion exchange materialis prepared in, or equilibrated to, the desired starting buffer. Uponpreparation, or equilibration, a slurry of the ion exchange material isobtained. The antibody solution is contacted with the slurry to adsorbthe antibody to be separated to the ion exchange material. The solutioncomprising the HCP(s) that do not bind to the ion exchange material isseparated from the slurry, e.g., by allowing the slurry to settle andremoving the supernatant. The slurry can be subjected to one or morewash steps. If desired, the slurry can be contacted with a solution ofhigher conductivity to desorb HCPs that have bound to the ion exchangematerial. In order to elute bound polypeptides, the salt concentrationof the buffer can be increased.

Ion exchange chromatography may also be used as an ion exchangeseparation technique. Ion exchange chromatography separates moleculesbased on differences between the overall charge of the molecules. Forthe purification of an antibody, the antibody must have a chargeopposite to that of the functional group attached to the ion exchangematerial, e.g., resin, in order to bind. For example, antibodies, whichgenerally have an overall positive charge in the buffer pH below its pI,will bind well to cation exchange material, which contain negativelycharged functional groups.

In ion exchange chromatography, charged patches on the surface of thesolute are attracted by opposite charges attached to a chromatographymatrix, provided the ionic strength of the surrounding buffer is low.Elution is generally achieved by increasing the ionic strength (i.e.,conductivity) of the buffer to compete with the solute for the chargedsites of the ion exchange matrix. Changing the pH and thereby alteringthe charge of the solute is another way to achieve elution of thesolute. The change in conductivity or pH may be gradual (gradientelution) or stepwise (step elution).

Anionic or cationic substituents may be attached to matrices in order toform anionic or cationic supports for chromatography. Non-limitingexamples of anionic exchange substituents include diethylaminoethyl(DEAE), quaternary aminoethyl(QAE) and quaternary amine(Q) groups.Cationic substitutents include carboxymethyl (CM), sulfoethyl(SE),sulfopropyl(SP), phosphate(P) and sulfonate(S). Cellulose ion exchangeresins such as DE23™, DE32™, DE52™, CM-23™, CM-32™, and CM-52™ areavailable from Whatman Ltd. Maidstone, Kent, U.K. SEPHADEX®-based and-locross-linked ion exchangers are also known. For example, DEAE-, QAE-,CM and SP-SEPHADEX® and DEAE Q CM- and S-SEPHAROSE® and SEPHAROSE® FastFlow are all available from Pharmacia AB. Further, both DEAE and CMderivitized ethylene glycol-methacrylate copolymer such as TOYOPEARL™DEAE-650S or M and TOYOPEARL™ CM-650S or M are available from Toso HaasCo., Philadelphia, Pa.

A mixture comprising an antibody and impurities, e.g., HCP(s), is loadedonto an ion exchange column, such as a cation exchange column. Forexample, but not by way of limitation, the mixture can be loaded at aload of about 80 g protein/L resin depending upon the column used. Anexample of a suitable cation exchange column is a 80 cm diameter×23 cmlong column whose bed volume is about 116 L. The mixture loaded ontothis cation column can subsequently washed with wash buffer(equilibration buffer). The antibody is then eluted from the column, anda first eluate is obtained.

This ion exchange step facilitates the capture of the antibody ofinterest while reducing impurities such as HCPs. In certain aspects, theion exchange column is a cation exchange column. For example, but not byway of limitation, a suitable resin for such a cation exchange column isCM HyperDF resin. These resins are available from commercial sourcessuch as Pall Corporation. This cation exchange procedure can be carriedout at or around room temperature.

4.5 Ultrafiltration/Diafiltration

Certain embodiments of the present invention employ ultrafiltrationand/or diafiltration steps to further purify and concentrate theantibody sample. Ultrafiltration is described in detail in:Microfiltration and Ultrafiltration: Principles and Applications, L.Zeman and A. Zydney (Marcel Dekker, Inc., New York, N.Y., 1996); and in:Ultrafiltration Handbook, Munir Cheryan (Technomic Publishing, 1986;ISBN No. 87762-456-9). A preferred filtration process is Tangential FlowFiltration as described in the Millipore catalogue entitled“Pharmaceutical Process Filtration Catalogue” pp. 177-202 (Bedford,Mass., 1995/96). Ultrafiltration is generally considered to meanfiltration using filters with a pore size of smaller than 0.1 μm. Byemploying filters having such small pore size, the volume of the samplecan be reduced through permeation of the sample buffer through thefilter while antibodies are retained behind the filter.

Diafiltration is a method of using ultrafilters to remove and exchangesalts, sugars, and non-aqueous solvents, to separate free from boundspecies, to remove low molecular-weight material, and/or to cause therapid change of ionic and/or pH environments. Microsolutes are removedmost efficiently by adding solvent to the solution being ultrafilteredat a rate approximately equal to the ultratfiltration rate. This washesmicrospecies from the solution at a constant volume, effectivelypurifying the retained antibody. In certain embodiments of the presentinvention, a diafiltration step is employed to exchange the variousbuffers used in connection with the instant invention, optionally priorto further chromatography or other purification steps, as well as toremove impurities from the antibody preparations.

4.6 Hydrophobic Interaction Chromatography

The present invention also features methods for producing a HCP-reducedantibody preparation from a mixture comprising an antibody and at leastone HCP further comprising a hydrophobic interaction separation step.For example, a first eluate obtained from an ion exchange column can besubjected to a hydrophobic interaction material such that a secondeluate having a reduced level of HCP is obtained. Hydrophobicinteraction chromatography steps, such as those disclosed herein, aregenerally performed to remove protein aggregates, such as antibodyaggregates, and process-related impurities.

In performing the separation, the sample mixture is contacted with theHIC material, e.g., using a batch purification technique or using acolumn. Prior to HIC purification it may be desirable to remove anychaotropic agents or very hydrophobic substances, e.g., by passing themixture through a pre-column.

For example, in the context of batch purification, HIC material isprepared in or equilibrated to the desired equilibration buffer. Aslurry of the HIC material is obtained. The antibody solution iscontacted with the slurry to adsorb the antibody to be separated to theHIC material. The solution comprising the HCPs that do not bind to theHIC material is separated from the slurry, e.g., by allowing the slurryto settle and removing the supernatant. The slurry can be subjected toone or more washing steps. If desired, the slurry can be contacted witha solution of lower conductivity to desorb antibodies that have bound tothe HIC material. In order to elute bound antibodies, the saltconcentration can be decreased.

Whereas ion exchange chromatography relies on the charges of theantibodies to isolate them, hydrophobic interaction chromatography usesthe hydrophobic properties of the antibodies. Hydrophobic groups on theantibody interact with hydrophobic groups on the column. The morehydrophobic a protein is the stronger it will interact with the column.Thus the HIC step removes host cell derived impurities (e.g., DNA andother high and low molecular weight product-related species).

Hydrophobic interactions are strongest at high ionic strength,therefore, this form of separation is conveniently performed followingsalt precipitations or ion exchange procedures. Adsorption of theantibody to a HIC column is favored by high salt concentrations, but theactual concentrations can vary over a wide range depending on the natureof the antibody and the particular HIC ligand chosen. Various ions canbe arranged in a so-called soluphobic series depending on whether theypromote hydrophobic interactions (salting-out effects) or disrupt thestructure of water (chaotropic effect) and lead to the weakening of thehydrophobic interaction. Cations are ranked in terms of increasingsalting out effect as Ba++; Ca++; Mg++; Li+; Cs+; Na+K+; Rb+; NH4+,while anions may be ranked in terms of increasing chaotropic effect asPO—; SO₄—; CH3CO3—; Cl—; Br—; NO3—; ClO4—; I—; SCN—.

In general, Na, K or NH4 sulfates effectively promote ligand-proteininteraction in HIC. Salts may be formulated that influence the strengthof the interaction as given by the following relationship:(NH4)2SO4>Na2SO4>NaCl>NH4Cl>NaBr>NaSCN. In general, salt concentrationsof between about 0.75 and about 2 M ammonium sulfate or between about 1and 4 M NaCl are useful.

HIC columns normally comprise a base matrix (e.g., cross-linked agaroseor synthetic copolymer material) to which hydrobobic ligands (e.g.,alkyl or aryl groups) are coupled. A suitable HIC column comprises anagarose resin substituted with phenyl groups (e.g., a Phenyl Sepharose™column). Many HIC columns are available commercially. Examples include,but are not limited to, Phenyl Sepharose™ 6 Fast Flow column with low orhigh substitution (Pharmacia LKB Biotechnology, AB, Sweden); PhenylSepharose™ High Performance column (Pharmacia LKB Biotechnology, AB,Sweden); Octyl Sepharose™ High Performance column (Pharmacia LKBBiotechnology, AB, Sweden); Fractogel™ EMD Propyl or Fractogel™ EMDPhenyl columns (E. Merck, Germany); Macro-Prep™ Methyl or Macro-Prep™t-Butyl Supports (Bio-Rad, California); WP HI-Propyl (C3)™ column (J. T.Baker, New Jersey); and Toyopearl™ ether, phenyl or butyl columns(TosoHaas, PA)

4.7 Exemplary Purification Strategies

In certain embodiments, primary recovery can proceed by sequentiallyemploying pH reduction, centrifugation, and filtration steps to removecells and cell debris (including HCPs) from the production bioreactorharvest. For example, but not by way of limitation, a culture comprisingantibodies, media, and cells can be subjected to pH-mediated virusreduction/inactivation using an acid pH of about 3.5 for approximately 1hour. The pH reduction can be facilitated using known acid preparationssuch as citric acid, e.g., 3 M citric acid. Exposure to acid pH reduces,if not completely eliminates, pH sensitive viral contaminants andprecipitates some media/cell contaminants. Following this viralreduction/inactivation step, the pH is adjusted to about 4.9 or 5.0using a base such as sodium hydroxide, e.g., 3 M sodium hydroxide, forabout twenty to about forty minutes. This adjustment can occur at around20° C. In certain embodiments, the pH adjusted culture then centrifugedat approximately 7000×g to approximately 11,000×g. In certainembodiments, the resulting sample supernatant is then passed through afilter train comprising multiple depth filters. In certain embodiments,the filter train comprises around twelve 16-inch Cuno™ model 30/60ZAdepth filters (3M Corp.) and around three round filter housings fittedwith three 30-inch 0.45/0.2 μm Sartopore™ 2 filter cartridges(Sartorius). The clarified supernatant is collected in a vessel such asa pre-sterilized harvest vessel and held at approximately 8° C. Thistemperature is then adjusted to approximately 20° C. prior to thecapture chromatography step or steps outlined below. It should be notedthat one skilled in the art may vary the conditions recited above andstill be within the scope of the present invention.

In certain embodiments, primary recovery will be followed by affinitychromatography using Protein A resin. There are several commercialsources for Protein A resin. One suitable resin is MabSelect™ from GEHealthcare. An example of a suitable column packed with MabSelect™ is acolumn about 1.0 cm diameter×about 21.6 cm long (˜17 mL bed volume).This size column can be used for bench scale. This can be compared withother columns used for scale ups. For example, a 20 cm×21 cm columnwhose bed volume is about 6.6 L can be used for commercial production.Regardless of the column, the column can be packed using a suitableresin such as MabSelect™

In certain aspects, the Protein A column can be equilibrated with asuitable buffer prior to sample loading. An example of a suitable bufferis a Tris/NaCl buffer, pH of about 6 to 8, preferably about 7.2. Aspecific example of suitable conditions is 25 mM Tris, 100 mM NaCl, pH7.2. Following this equilibration, the sample can be loaded onto thecolumn. Following the loading of the column, the column can be washedone or multiple times using, e.g., the equilibrating buffer. Otherwashes including washes employing different buffers can be used beforeeluting the column. For example, the column can be washed using one ormore column volumes of 20 mM citric acid/sodium citrate, 0.5 M NaCl atpH of about 6.0. This wash can optionally be followed by one or morewashes using the equilibrating buffer. The Protein A column can then beeluted using an appropriate elution buffer. An example of a suitableelution buffer is an acetic acid/NaCl buffer, pH around 3.5. Suitableconditions are, e.g., 0.1 M acetic acid, pH 3.5. The eluate can bemonitored using techniques well known to those skilled in the art. Forexample, the absorbance at OD₂₈₀ can be followed. Column eluate can becollected starting with an initial deflection of about 0.5 AU to areading of about 0.5 AU at the trailing edge of the elution peak. Theelution fraction(s) of interest can then be prepared for furtherprocessing. For example, the collected sample can be titrated to a pH ofabout 5.0 using Tris (e.g., 1.0 M) at a pH of about 10. Optionally, thistitrated sample can be filtered and further processed.

The dynamic binding capacity (DBC) of the MabSelect™ column can bedetermined either by a single flow rate load or dual-flow load strategy.The single flow rate load can be evaluated at a velocity of about 300cm/hr throughout the entire loading period. The dual-flow rate loadstrategy can be determined by loading the column up to about 35 mgprotein/mL resin at a linear velocity of about 300 cm/hr, then reducingthe linear velocity by half to allow longer residence time for the lastportion of the load.

The Protein A eluate can then be further purified using a cationexchange column. In certain embodiments, the equilibrating buffer usedin the cation exchange column is a buffer having a pH of about 5.0. Anexample of a suitable buffer is about 210 mM sodium acetate, pH 5.0.Following equilibration, the column is loaded with sample prepared fromthe primary recovery step above. The column is packed with a cationexchange resin, such as CM Sepharose™ Fast Flow from GE Healthcare. Thecolumn is then washed using the equilibrating buffer. The column is nextsubjected to an elution step using a buffer having a greater ionicstrength as compared to the equilibrating or wash buffer. For example, asuitable elution buffer can be about 790 mM sodium acetate, pH 5.0. Theantibodies will be eluted and can be monitored using a UVspectrophotometer set at OD_(280nm). In a particular example, elutioncollection can be from upside 3 OD_(280nm) to downside 8 OD_(280nm). Itshould be understood that one skilled in the art may vary the conditionsand yet still be within the scope of the invention.

In certain embodiments the Protein A eluate is instead further purifiedusing an anion exchange column. A non-limiting example of a suitablecolumn for this step is a 60 cm diameter×30 cm long column whose bedvolume is about 85 L. The column is packed with an anion exchange resin,such as Q Sepharose™ Fast Flow from GE Healthcare. The column can beequilibrated using about seven column volumes of an appropriate buffersuch as Tris/sodium chloride. An example of suitable conditions are 25mM Tris, 50 mM sodium chloride at pH 8.0. A skilled artisan may vary theconditions but still be within the scope of the present invention. Thecolumn is loaded with the collected sample from the Protein Apurification step outlined above. In another aspect, the column isloaded from the eluate collected during cation exchange. Following theloading of the column, the column is washed with the equilibrationbuffer (e.g., the Tris/sodium chloride buffer). The flow-throughcomprising the antibodies can be monitored using a UV spectrophotometerat OD_(280nm). This anion exchange step reduces process relatedimpurities such as nucleic acids like DNA, and host cell proteins. Theseparation occurs due to the fact that the antibodies of interest do notsubstantially interact with nor bind to the solid phase of the column,e.g., to the Q Sepharose™, but many impurities do interact with and bindto the column's solid phase. The anion exchange can be performed atabout 12° C.

In certain embodiments, the cation exchange or anion exchange eluate,depending on which ion exchange step is employed first, is next filteredusing, e.g., a 16 inch Cuno™ delipid filter. This filtration, using thedelipid filter, can be followed by, e.g., a 30-inch 0.45/0.2 μmSartopore™ bi-layer filter cartridge. The ion exchange elution buffercan be used to flush the residual volume remaining in the filters andprepared for ultrafiltration/diafiltration.

In order to accomplish the ultratfiltration/diafiltration step, thefiltration media is prepared in a suitable buffer, e.g., 20 mM sodiumphosphate, pH 7.0. A salt such as sodium chloride can be added toincrease the ionic strength, e.g., 100 mM sodium chloride. Thisultrafiltration/diafiltration step serves to concentrate the anti-IL-12,anti-TNFα, or anti-IL-18 antibodies, remove the sodium acetate andadjust the pH. Commercial filters are available to effectuate this step.For example, Millipore manufactures a 30 kD molecular weight cut-off(MWCO) cellulose ultrafilter membrane cassette. This filtrationprocedure can be conducted at or around room temperature.

In certain embodiments, the sample from the capture filtration stepabove is subjected to a second ion exchange separation step. Preferablythis second ion exchange separation will involve separation based on theopposite charge of the first ion exchange separation. For example, if ananion exchange step is employed after primary recovery, the second ionexchange chromatographic step may be a cation exchange step. Conversely,if the primary recovery step was followed by a cation exchange step,that step would be followed by an anion exchange step. In certainembodiments the first ion exchange eluate can be subjected directly tothe second ion exchange chromatographic step where the first ionexchange eluate is adjusted to the appropriate buffer conditions.Suitable anionic and cationic separation materials and conditions aredescribed above.

In certain embodiments of the instant invention the sample containingantibodies will be further processed using a hydrophobic interactionseparation step. A non-limiting example of a suitable column for such astep is an 80 cm diameter×15 cm long column whose bed volume is about 75L, which is packed with an appropriate resin used for HIC such as, butnot limited to, Phenyl HP Sepharose™ from Amersham Biosciences, Upsala,Sweden. The flow-through preparation obtained from the previous anionexchange chromatography step comprising the antibodies of interest canbe diluted with an equal volume of around 1.7 M ammonium sulfate, 50 mMsodium phosphate, pH 7.0. This then can be subjected to filtration usinga 0.45/0.2 μm Sartopore™ 2 bi-layer filter, or its equivalent. Incertain embodiments, the hydrophobic chromatography procedure involvestwo or more cycles.

In certain embodiments, the HIC column is first equilibrated using asuitable buffer. A non-limiting example of a suitable buffer is 0.85 Mammonium sulfate, 50 mM sodium phosphate, pH 7.0. One skilled in the artcan vary the equilibrating buffer and still be within the scope of thepresent invention by altering the concentrations of the buffering agentsand/or by substituting equivalent buffers. In certain embodiments thecolumn is then loaded with an anion exchange flow-through sample andwashed multiple times, e.g., three times, with an appropriate buffersystem such as ammonium sulfate/sodium phosphate. An example of asuitable buffer system includes 1.1 M ammonium sulfate, 50 mM sodiumphosphate buffer with a pH of around 7.0. Optionally, the column canundergo further wash cycles. For example, a second wash cycle caninclude multiple column washes, e.g., one to seven times, using anappropriate buffer system. A non-limiting example of a suitable buffersystem includes 0.85 M ammonium sulfate, 50 mM sodium phosphate, pH 7.0.In one aspect, the loaded column undergoes yet a third wash using anappropriate buffer system. The column can be washed multiple times,e.g., one to three times, using a buffer system such as 1.1 M ammoniumsulfate, 50 mM sodium phosphate at a pH around 7.0. Again, one skilledin the art can vary the buffering conditions and still be within thescope of the present invention.

The column is eluted using an appropriate elution buffer. A suitableexample of such an elution buffer is 0.5 M ammonium sulfate, 15 mMsodium phosphate at a pH around 7.0. The antibodies of interest can bedetected and collected using a conventional spectrophotometer from theupside at 3 OD_(280 nm) to downside of peak at 3 OD_(280 nm).

In certain aspects of the invention, the eluate from the hydrophobicchromatography step is subjected to filtration for the removal of viralparticles, including intact viruses, if present. A non-limiting exampleof a suitable filter is the Ultipor DV50™ filter from Pall Corporation.Other viral filters can be used in this filtration step and are wellknown to those skilled in the art. The HIC eluate is passed through apre-wetted filter of about 0.1 μm and a 2×30-inch Ultipor DV50™ filtertrain at around 34 psig. In certain embodiments, following thefiltration process, the filter is washed using, e.g., the HIC elutionbuffer in order to remove any antibodies retained in the filter housing.The filtrate can be stored in a pre-sterilized container at around 12°C.

In a certain embodiments, the filtrate from the above is again subjectedto ultrafiltration/diafiltration. This step is important if apractitioner's end point is to use the antibody in a, e.g.,pharmaceutical formulation. This process, if employed, can facilitatethe concentration of antibody, removal of buffering salts previouslyused and replace it with a particular formulation buffer. In certainembodiments, continuous diafiltration with multiple volumes, e.g., twovolumes, of a formulation buffer is performed. A non-limiting example ofa suitable formulation buffer is 5 mM methionine, 2% mannitol, 0.5%sucrose, pH 5.9 buffer (no Tween). Upon completion of this diavolumeexchange the antibodies are concentrated. Once a predeterminedconcentration of antibody has been achieved, then a practitioner cancalculate the amount of 10% Tween that should be added to arrive at afinal Tween concentration of about 0.005% (v/v).

Certain embodiments of the present invention will include furtherpurification steps. Examples of additional purification procedures whichcan be performed prior to, during, or following the ion exchangechromatography method include ethanol precipitation, isoelectricfocusing, reverse phase HPLC, chromatography on silica, chromatographyon heparin Sepharose™, further anion exchange chromatography and/orfurther cation exchange chromatography, chromatofocusing, SDS-PAGE,ammonium sulfate precipitation, hydroxylapatite chromatography, gelelectrophoresis, dialysis, and affinity chromatography (e.g., usingprotein G, an antibody, a specific substrate, ligand or antigen as thecapture reagent).

In certain embodiments of the present invention, the anti-IL-12 antibodyis an IgA₁, IgA₂, IgD, IgE, IgG₁, IgG₂, IgG₃, IgG₄, or IgM isotypeantibody comprising the heavy and light chain variable region sequencesoutlined in FIG. 1. In preferred embodiments, the anti-IL-12 antibody isan IgG₁, IgG₂, IgG₃ or IgG₄ isotype antibody comprising the heavy andlight chain variable region sequences outlined in FIG. 1, morepreferably the anti-IL-12 antibody is an IgG₁ antibody comprising theheavy and light chain variable region sequences outlined in FIG. 1. Incertain embodiments of the present invention, the anti-TNFα antibody isan IgA₁, IgA₂, IgD, IgE, IgG₁, IgG₂, IgG₃, IgG₄, or IgM isotype antibodycomprising the heavy and light chain variable region sequences outlinedin FIG. 3. In preferred embodiments, the anti-TNFα antibody is an IgG₁,IgG₂, IgG₃ or IgG₄ isotype antibody comprising the heavy and light chainvariable region sequences outlined in FIG. 3, more preferably theanti-TNFα antibody is an IgG₁ antibody comprising the heavy and lightchain variable region sequences outlined in FIG. 3.

5. Methods of Assaying Sample Purity

5.1 Assaying Host Cell Protein

The present invention also provides methods for determining the residuallevels of host cell protein (HCP) concentration in the isolated/purifiedantibody composition. As described above, HCPs are desirably excludedfrom the final target substance product, e.g., the anti-IL-12,anti-TNFα, or anti-IL-18 antibody. Exemplary HCPs include proteinsoriginating from the source of the antibody production. Failure toidentify and sufficiently remove HCPs from the target antibody may leadto reduced efficacy and/or adverse subject reactions.

As used herein, the term “HCP ELISA” refers to an ELISA where the secondantibody used in the assay is specific to the HCPs produced from cells,e.g., CHO cells, used to generate the antibody (e.g., anti-IL-12,anti-TNFα, or anti-IL-18 antibody). The second antibody may be producedaccording to conventional methods known to those of skill in the art.For example, the second antibody may be produced using HCPs obtained bysham production and purification runs, i.e., the same cell line used toproduce the antibody of interest is used, but the cell line is nottransfected with antibody DNA. In an exemplary embodiment, the secondantibody is produced using HPCs similar to those expressed in the cellexpression system of choice, i.e., the cell expression system used toproduce the target antibody.

Generally, HCP ELISA comprises sandwiching a liquid sample comprisingHCPs between two layers of antibodies, i.e., a first antibody and asecond antibody. The sample is incubated during which time the HCPs inthe sample are captured by the first antibody, for example, but notlimited to goat anti-CHO, affinity purified (Cygnus). A labeled secondantibody, or blend of antibodies, specific to the HCPs produced from thecells used to generate the antibody, e.g., anti-CHO HCP Biotinylated, isadded, and binds to the HCPs within the sample. In certain embodimentsthe first and second antibodies are polyclonal antibodies. In certainaspects the first and second antibodies are blends of polyclonalantibodies raised against HCPs, for example, but not limited toBiotinylated goat anti Host Cell Protein Mixture 599/626/748. The amountof HCP contained in the sample is determined using the appropriate testbased on the label of the second antibody.

HCP ELISA may be used for determining the level of HCPs in an antibodycomposition, such as an eluate or flow-through obtained using theprocess described above. The present invention also provides acomposition comprising an antibody, wherein the composition has nodetectable level of HCPs as determined by an HCP Enzyme LinkedImmunosorbent Assay (“ELISA”).

5.2 Assaying Affinity Chromatographic Material

In certain embodiments, the present invention also provides methods fordetermining the residual levels of affinity chromatographic material inthe isolated/purified antibody composition. In certain contexts suchmaterial leaches into the antibody composition during the purificationprocess. In certain embodiments, an assay for identifying theconcentration of Protein A in the isolated/purified antibody compositionis employed. As used herein, the term “Protein A ELISA” refers to anELISA where the second antibody used in the assay is specific to theProtein A employed to purify the antibody of interest, e.g., ananti-IL-12, anti-TNFα, or anti-IL-18 antibody. The second antibody maybe produced according to conventional methods known to those of skill inthe art. For example, the second antibody may be produced usingnaturally occurring or recombinant Protein A in the context ofconventional methods for antibody generation and production.

Generally, Protein A ELISA comprises sandwiching a liquid samplecomprising Protein A (or possibly containing Protein A) between twolayers of anti-Protein A antibodies, i.e., a first anti-Protein Aantibody and a second anti-Protein A antibody. The sample is exosed to afirst layer of anti-Protein A antibody, for example, but not limited topolyclonal antibodies or blends of polyclonal antibodies, and incubatedfor a time sufficient for Protein A in the sample to be captured by thefirst antibody. A labeled second antibody, for example, but not limitedto polyclonal antibodies or blends of polyclonal antibodies, specific tothe Protein A is then added, and binds to the captured Protein A withinthe sample. Additional non-limiting examples of anti-Protein Aantibodies useful in the context of the instant invention includechicken anti-Protein A and biotinylated anti-Protein A antibodies. Theamount of Protein A contained in the sample is determined using theappropriate test based on the label of the second antibody. Similarassays can be employed to identify the concentration of alternativeaffinity chromatographic materials.

Protein A ELISA may be used for determining the level of Protein A in anantibody composition, such as an eluate or flow-through obtained usingthe process described in above. The present invention also provides acomposition comprising an antibody, wherein the composition has nodetectable level of Protein A as determined by an Protein A EnzymeLinked Immunosorbent Assay (“ELISA”).

6. Further Modifications

The antibodies of the present invention can be modified. In someembodiments, the antibodies or antigen binding fragments thereof arechemically modified to provide a desired effect. For example, pegylationof antibodies or antibody fragments of the invention may be carried outby any of the pegylation reactions known in the art, as described, e.g.,in the following references: Focus on Growth Factors 3:4-10 (1992); EP 0154 316; and EP 0 401 384, each of which is incorporated by referenceherein in its entirety. In one aspect, the pegylation is carried out viaan acylation reaction or an alkylation reaction with a reactivepolyethylene glycol molecule (or an analogous reactive water-solublepolymer). A suitable water-soluble polymer for pegylation of theantibodies and antibody fragments of the invention is polyethyleneglycol (PEG). As used herein, “polyethylene glycol” is meant toencompass any of the forms of PEG that have been used to derivatizeother proteins, such as mono (Cl—ClO) alkoxy- or aryloxy-polyethyleneglycol.

Methods for preparing pegylated antibodies and antibody fragments of theinvention will generally comprise the steps of (a) reacting the antibodyor antibody fragment with polyethylene glycol, such as a reactive esteror aldehyde derivative of PEG, under suitable conditions whereby theantibody or antibody fragment becomes attached to one or more PEGgroups, and (b) obtaining the reaction products. It will be apparent toone of ordinary skill in the art to select the optimal reactionconditions or the acylation reactions based on known parameters and thedesired result.

Pegylated antibodies and antibody fragments specific for IL-12, TNFα, orIL-18 may generally be used to treat IL-12-related, TNFα-related, orIL-18-related disorders of the invention by administration of theanti-IL-12, anti-TNFα or anti-IL-18 antibodies and antibody fragmentsdescribed herein. Generally the pegylated antibodies and antibodyfragments have increased half-life, as compared to the nonpegylatedantibodies and antibody fragments. The pegylated antibodies and antibodyfragments may be employed alone, together, or in combination with otherpharmaceutical compositions.

An antibody or antibody portion of the invention can be derivatized orlinked to another functional molecule (e.g., another peptide orprotein). Accordingly, the antibodies and antibody portions of theinvention are intended to include derivatized and otherwise modifiedforms of the human anti-hIL-12, anti-TNFα, or anti-hIL-18 antibodiesdescribed herein, including immunoadhesion molecules. For example, anantibody or antibody portion of the invention can be functionally linked(by chemical coupling, genetic fusion, noncovalent association orotherwise) to one or more other molecular entities, such as anotherantibody (e.g., a bispecific antibody or a diabody), a detectable agent,a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptidethat can mediate associate of the antibody or antibody portion withanother molecule (such as a streptavidin core region or a polyhistidinetag).

One type of derivatized antibody is produced by crosslinking two or moreantibodies (of the same type or of different types, e.g., to createbispecific antibodies). Suitable crosslinkers include those that areheterobifunctional, having two distinctly reactive groups separated byan appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkersare available from Pierce Chemical Company, Rockford, Ill.

Useful detectable agents with which an antibody or antibody portion ofthe invention may be derivatized include fluorescent compounds.Exemplary fluorescent detectable agents include fluorescein, fluoresceinisothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonylchloride, phycoerythrin and the like. An antibody may also bederivatized with detectable enzymes, such as alkaline phosphatase,horseradish peroxidase, glucose oxidase and the like. When an antibodyis derivatized with a detectable enzyme, it is detected by addingadditional reagents that the enzyme uses to produce a detectablereaction product. For example, when the detectable agent horseradishperoxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody may also be derivatized with biotin, anddetected through indirect measurement of avidin or streptavidin binding.

7. Pharmaceutical Compositions

The antibodies and antibody-portions of the invention can beincorporated into pharmaceutical compositions suitable foradministration to a subject. Typically, the pharmaceutical compositioncomprises an antibody or antibody portion of the invention and apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible.Examples of pharmaceutically acceptable carriers include one or more ofwater, saline, phosphate buffered saline, dextrose, glycerol, ethanoland the like, as well as combinations thereof. In many cases, it isdesirable to include isotonic agents, e.g., sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition.Pharmaceutically acceptable carriers may further comprise minor amountsof auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the antibody or antibody portion.

The antibodies and antibody-portions of the invention can beincorporated into a pharmaceutical composition suitable for parenteraladministration. The antibody or antibody-portions can be prepared as aninjectable solution containing, e.g., 0.1-250 mg/mL antibody. Theinjectable solution can be composed of either a liquid or lyophilizeddosage form in a flint or amber vial, ampule or pre-filled syringe. Thebuffer can be L-histidine approximately 1-50 mM, (optimally 5-10 mM), atpH 5.0 to 7.0 (optimally pH 6.0). Other suitable buffers include but arenot limited to sodium succinate, sodium citrate, sodium phosphate orpotassium phosphate. Sodium chloride can be used to modify the toxicityof the solution at a concentration of 0-300 mM (optimally 150 mM for aliquid dosage form). Cryoprotectants can be included for a lyophilizeddosage form, principally 0-10% sucrose (optimally 0.5-1.0%). Othersuitable cryoprotectants include trehalose and lactose. Bulking agentscan be included for a lyophilized dosage form, principally 1-10%mannitol (optimally 24%). Stabilizers can be used in both liquid andlyophilized dosage forms, principally 1-50 mM L-methionine (optimally5-10 mM). Other suitable bulking agents include glycine, arginine, canbe included as 0-0.05% polysorbate-80 (optimally 0.005-0.01%).Additional surfactants include but are not limited to polysorbate 20 andBRIJ surfactants.

In one aspect, the pharmaceutical composition includes the antibody at adosage of about 0.01 mg/kg-10 mg/kg. In another aspect, the dosages ofthe antibody include approximately 1 mg/kg administered every otherweek, or approximately 0.3 mg/kg administered weekly. A skilledpractitioner can ascertain the proper dosage and regime foradministering to a subject.

The compositions of this invention may be in a variety of forms. Theseinclude, e.g., liquid, semi-solid and solid dosage forms, such as liquidsolutions (e.g., injectable and infusible solutions), dispersions orsuspensions, tablets, pills, powders, liposomes and suppositories. Theform depends on, e.g., the intended mode of administration andtherapeutic application. Typical compositions are in the form ofinjectable or infusible solutions, such as compositions similar to thoseused for passive immunization of humans with other antibodies. One modeof administration is parenteral (e.g., intravenous, subcutaneous,intraperitoneal, intramuscular). In one aspect, the antibody isadministered by intravenous infusion or injection. In another aspect,the antibody is administered by intramuscular or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (i.e.,antibody or antibody portion) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile,lyophilized powders for the preparation of sterile injectable solutions,the methods of preparation are vacuum drying and spray-drying thatyields a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof. Theproper fluidity of a solution can be maintained, e.g., by the use of acoating such as lecithin, by the maintenance of the required particlesize in the case of dispersion and by the use of surfactants. Prolongedabsorption of injectable compositions can be brought about by includingin the composition an agent that delays absorption, e.g., monostearatesalts and gelatin.

The antibodies and antibody-portions of the present invention can beadministered by a variety of methods known in the art, one route/mode ofadministration is subcutaneous injection, intravenous injection orinfusion. As will be appreciated by the skilled artisan, the routeand/or mode of administration will vary depending upon the desiredresults. In certain embodiments, the active compound may be preparedwith a carrier that will protect the compound against rapid release,such as a controlled release formulation, including implants,transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978, theentire teaching of which is incorporated herein by reference.

In certain aspects, an antibody or antibody portion of the invention maybe orally administered, e.g., with an inert diluent or an assimilableedible carrier. The compound (and other ingredients, if desired) mayalso be enclosed in a hard or soft shell gelatin capsule, compressedinto tablets, or incorporated directly into the subject's diet. For oraltherapeutic administration, the compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.To administer a compound of the invention by other than parenteraladministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.

Supplementary active compounds can also be incorporated into thecompositions. In certain aspects, an antibody or antibody portion of theinvention is co-formulated with and/or co-administered with one or moreadditional therapeutic agents that are useful for treating disorders inwhich IL-12, TNFα, or IL-18 activity is detrimental. For example, ananti-hIL-12, anti-TNFα or anti-IL-18 antibody or antibody portion of theinvention may be co-formulated and/or co-administered with one or moreadditional antibodies that bind other targets (e.g., antibodies thatbind other cytokines or that bind cell surface molecules). Furthermore,one or more antibodies of the invention may be used in combination withtwo or more of the foregoing therapeutic agents. Such combinationtherapies may advantageously utilize lower dosages of the administeredtherapeutic agents, thus avoiding possible toxicities or complicationsassociated with the various monotherapies. It will be appreciated by theskilled practitioner that when the antibodies of the invention are usedas part of a combination therapy, a lower dosage of antibody may bedesirable than when the antibody alone is administered to a subject(e.g., a synergistic therapeutic effect may be achieved through the useof combination therapy which, in turn, permits use of a lower dose ofthe antibody to achieve the desired therapeutic effect).

It should be understood that the antibodies of the invention or antigenbinding portion thereof can be used alone or in combination with anadditional agent, e.g., a therapeutic agent, said additional agent beingselected by the skilled artisan for its intended purpose. For example,the additional agent can be a therapeutic agent art-recognized as beinguseful to treat the disease or condition being treated by the antibodyof the present invention. The additional agent also can be an agentwhich imparts a beneficial attribute to the therapeutic composition,e.g., an agent which effects the viscosity of the composition.

It should further be understood that the combinations which are to beincluded within this invention are those combinations useful for theirintended purpose. The agents set forth below are illustrative and notintended to be limited. The combinations which are part of thisinvention can be the antibodies of the present invention and at leastone additional agent selected from the lists below. The combination canalso include more than one additional agent, e.g., two or threeadditional agents if the combination is such that the formed compositioncan perform its intended function.

Some combinations are non-steroidal anti-inflammatory drug(s) alsoreferred to as NSAIDS which include drugs like ibuprofen. Othercombinations are corticosteroids including prednisolone; the well knownside-effects of steroid use can be reduced or even eliminated bytapering the steroid dose required when treating patients in combinationwith the antibodies of this invention. Non-limiting examples oftherapeutic agents for rheumatoid arthritis with which an antibody, orantibody portion, of the invention can be combined to include thefollowing: cytokine suppressive anti-inflammatory drug(s) (CSAIDs);antibodies to or antagonists of other human cytokines or growth factors,for example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18,EMAP-II, GM-CSF, FGF, and PDGF. Antibodies of the invention, or antigenbinding portions thereof, can be combined with antibodies to cellsurface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40,CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, or their ligands includingCD 154 (gp39 or CD40L).

Some combinations of therapeutic agents may interfere at differentpoints in the autoimmune and subsequent inflammatory cascade; examplesinclude TNF antagonists like chimeric, humanized or human TNFantibodies, D2E7, (U.S. application Ser. No. 08/599,226 filed Feb. 9,1996, the entire teaching of which is incorporated herein by reference),cA2 (Remicade™), CDP 571, anti-TNF antibody fragments (e.g., CDP870),and soluble p55 or p75 TNF receptors, derivatives thereof, (p75TNFRIgG(Enbrel™) or p55TNFR1gG (Lenercept), soluble IL-13 receptor (sIL-13),and also TNFα converting enzyme (TACE) inhibitors; similarly IL-1inhibitors (e.g., Interleukin-1-converting enzyme inhibitors, such asVx740, or IL-1RA, etc.) may be effective for the same reason. Othercombinations include Interleukin 11, anti-P7s and p-selectinglycoprotein ligand (PSGL). Yet other combinations involve other keyplayers of the autoimmune response which may act parallel to, dependenton or in concert with IL-12 function; especially included are IL-18antagonists including IL-18 antibodies or soluble IL-18 receptors, orIL-18 binding proteins. It has been shown that IL-12 and IL-18 haveoverlapping but distinct functions and a combination of antagonists toboth may be most effective. Yet another combination includesnon-depleting anti-CD4 inhibitors. Yet other combinations includeantagonists of the co-stimulatory pathway CD80 (B7.1) or CD86 (B7.2)including antibodies, soluble receptors or antagonistic ligands.

The antibodies of the invention, or antigen binding portions thereof,may also be combined with agents, such as methotrexate, 6-MP,azathioprine sulphasalazine, mesalazine, olsalazinechloroquinine/hydroxychloroquine, pencillamine, aurothiomalate(intramuscular and oral), azathioprine, cochicine, corticosteroids(oral, inhaled and local injection), β-2 adrenoreceptor agonists(salbutamol, terbutaline, salmeteral), xanthines (theophylline,aminophylline), cromoglycate, nedocromil, ketotifen, ipratropium andoxitropium, cyclosporin, FK506, rapamycin, mycophenolate mofetil,leflunomide, NSAIDs, for example, ibuprofen, corticosteroids such asprednisolone, phosphodiesterase inhibitors, adensosine agonists,antithrombotic agents, complement inhibitors, adrenergic agents, agentswhich interfere with signalling by proinflammatory cytokines such asTNFα or IL-1 (e.g., IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL-1βconverting enzyme inhibitors (e.g., Vx740), anti-P7s, p-selectinglycoprotein ligand (PSGL), TNFα converting enzyme (TACE) inhibitors,T-cell signaling inhibitors such as kinase inhibitors, metalloproteinaseinhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensinconverting enzyme inhibitors, soluble cytokine receptors and derivativesthereof (e.g., soluble p55 or p75 TNF receptors and the derivativesp75TNFRIgG (Enbrel™) and p55TNFRIgG (Lenercept), sIL-1 RI, sIL-1RII,sIL-6R, soluble IL-13 receptor (sIL-13)) and anti-inflammatory cytokines(e.g., IL-4, IL-10, IL-11, IL-13 and TGFβ). Some combinations includemethotrexate or leflunomide and in moderate or severe rheumatoidarthritis cases, cyclosporine.

Non-limiting examples of therapeutic agents for inflammatory boweldisease with which an antibody, or antibody portion, of the inventioncan be combined include the following: budenoside, epidermal growthfactor, corticosteroids, cyclosporin, sulfasalazine, aminosalicylates,6-mercaptopurine, azathioprine, metronidazole, lipoxygenase inhibitors,mesalamine, olsalazine, balsalazide, antioxidants, thromboxaneinhibitors, IL-1 receptor antagonists, anti-IL-1α monoclonal antibodies,anti-IL-6 monoclonal antibodies, growth factors, elastase inhibitors,pyridinyl-imidazole compounds, antibodies to or antagonists of otherhuman cytokines or growth factors, e.g., TNF, LT, IL-1, IL-2, IL-6,IL-7, IL-8, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF, and PDGF.Antibodies of the invention, or antigen binding portions thereof, can becombined with antibodies to cell surface molecules such as CD2, CD3,CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their ligands. Theantibodies of the invention, or antigen binding portions thereof, mayalso be combined with agents, such as methotrexate, cyclosporin, FK506,rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, e.g., ibuprofen,corticosteroids such as prednisolone, phosphodiesterase inhibitors,adenosine agonists, antithrombotic agents, complement inhibitors,adrenergic agents, agents which interfere with signaling byproinflammatory cytokines such as TNFα or IL-1 (e.g., IRAK, NIK, IKK,p38 or MAP kinase inhibitors), IL-1β converting enzyme inhibitors (e.g.,Vx740), anti-P7s, p-selectin glycoprotein ligand (PSGL), TNFα convertingenzyme inhibitors, T-cell signaling inhibitors such as kinaseinhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine,6-mercaptopurines, angiotensin converting enzyme inhibitors, solublecytokine receptors and derivatives thereof (e.g., soluble p55 or p75 TNFreceptors, sIL-1RI, sIL-1RII, sIL-6R, soluble IL-13 receptor (sIL-13))and anti-inflammatory cytokines (e.g., IL-4, IL-10, IL-11, IL-13 andTGFβ).

Examples of therapeutic agents for Crohn's disease in which an antibodyor an antigen binding portion can be combined include the following: TNFantagonists, e.g., anti-TNF antibodies, D2E7 (U.S. application Ser. No.08/599,226, filed Feb. 9, 1996, the entire teaching of which isincorporated herein by reference), cA2 (Remicade™), CDP 571, anti-TNFantibody fragments (e.g., CDP870), TNFR-Ig constructs(p75TNFRIgG(Enbrel™) and p55TNFRIgG (Lenercept)), anti-P7s, p-selectin glycoproteinligand (PSGL), soluble IL-13 receptor (sIL-13), and PDE4 inhibitors.Antibodies of the invention or antigen binding portions thereof, can becombined with corticosteroids, e.g., budenoside and dexamethasone.Antibodies of the invention or antigen binding portions thereof, mayalso be combined with agents such as sulfasalazine, 5-aminosalicylicacid and olsalazine, and agents which interfere with synthesis or actionof proinflammatory cytokines such as IL-1, e.g., IL-1 converting enzymeinhibitors (e.g., Vx740) and IL-lra. Antibodies of the invention orantigen binding portion thereof may also be used with T cell signalinginhibitors, e.g., tyrosine kinase inhibitors 6-mercaptopurines.Antibodies of the invention or antigen binding portions thereof, can becombined with IL-11.

Non-limiting examples of therapeutic agents for multiple sclerosis withwhich an antibody, or antibody portion, of the invention can be combinedinclude the following: corticosteroids, prednisolone,methylprednisolone, azathioprine, cyclophosphamide, cyclosporine,methotrexate, 4-aminopyridine, tizanidine, IFNβ1a (Avonex; Biogen),IFNβ1b (Betaseron; Chiron/Berlex), Copolymer 1 (Cop-1, Copaxone, TevaPharmaceutical Industries, Inc.), hyperbaric oxygen, intravenousimmunoglobulin, clabribine, antibodies to or antagonists of other humancytokines or growth factors, e.g., TNF, LT, IL-1, IL-2, IL-6, IL-7,IL-8, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF, and PDGF. Antibodies ofthe invention, or antigen binding portions thereof, can be combined withantibodies to cell surface molecules such as CD2, CD3, CD4, CD8, CD25,CD28, CD30, CD40, CD45, CD69, CD80, CD86, CD90 or their ligands. Theantibodies of the invention, or antigen binding portions thereof, mayalso be combined with agents, such as methotrexate, cyclosporine, FK506,rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, e.g., ibuprofen,corticosteroids such as prednisolone, phosphodiesterase inhibitors,adensosine agonists, antithrombotic agents, complement inhibitors,adrenergic agents, agents which interfere with signaling byproinflammatory cytokines such as TNFα or IL-1 (e.g., IRAK, NIK, IKK,p38 or MAP kinase inhibitors), IL-1β converting enzyme inhibitors (e.g.,Vx740), anti-P7s, p-selectin glycoprotein ligand (PSGL), TACEinhibitors, T-cell signaling inhibitors such as kinase inhibitors,metalloproteinase inhibitors, sulfasalazine, azathioprine,6-mercaptopurines, angiotensin converting enzyme inhibitors, solublecytokine receptors and derivatives thereof (e.g., soluble p55 or p75 TNFreceptors, sIL-1 RI, sIL-1 RII, sIL-6R, soluble IL-13 receptor (sIL-13))and anti-inflammatory cytokines (e.g., IL-4, IL-10, IL-13 and TGFβ).

Examples of therapeutic agents for multiple sclerosis in which theantibody or antigen binding portion thereof can be combined to includeIFNβ, e.g., IFNβ1a and IFNβ1b, copaxone, corticosteroids, IL-1inhibitors, TNF inhibitors, and antibodies to CD40 ligand and CD80.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody or antibody portion of the invention. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of the antibodyor antibody portion may vary according to factors such as the diseasestate, age, sex, and weight of the individual, and the ability of theantibody or antibody portion to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the antibody or antibody portion areoutweighed by the therapeutically beneficial effects. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. In certainembodiments it is especially advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit comprising a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic or prophylactic effect to be achieved, and(b) the limitations inherent in the art of compounding such an activecompound for the treatment of sensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody or antibody portion ofthe invention is 0.01-20 mg/kg, or 1-10 mg/kg, or 0.3-1 mg/kg. It is tobe noted that dosage values may vary with the type and severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

8. Uses of the Antibodies of the Invention

8.1. Anti-IL-12 Antibody Uses Generally

Given their ability to bind to IL-12, the anti-IL-12 antibodies, orportions thereof, of the invention can be used to detect IL-12, in oneaspect, hIL-12 (e.g., in a sample matrix, in one aspect, a biologicalsample, such as serum or plasma), using a conventional immunoassay, suchas an enzyme linked immunosorbent assays (ELISA), an radioimmunoassay(RIA) or tissue immunohistochemistry. The invention provides a methodfor detecting IL-12 in a biological sample comprising contacting asample with an antibody, or antibody portion, of the invention anddetecting either the antibody (or antibody portion) bound to IL-12 orunbound antibody (or antibody portion), to thereby detect IL-12 in thesample. The antibody is directly or indirectly labeled with a detectablesubstance to facilitate detection of the bound or unbound antibody.Suitable detectable substances include various enzymes, prostheticgroups, fluorescent materials, luminescent materials and radioactivematerials. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examplesof suitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; and examples ofsuitable radioactive material include 125 I, 131 I, 35 S, or 3H.Detection of IL-12 in a sample may be useful in a diagnostic context,for example in the diagnosis of a condition associated with increasedIL-12, and/or may be useful in identifying a subject who may benefitfrom treatment with an anti-IL-12 antibody.

Alternative to labeling the antibody, IL-12 can be assayed in a sampleby a competition immunoassay utilizing, e.g., rhIL-12 standards labeledwith a detectable substance and an unlabeled anti-IL-12 antibody, suchas an anti-hIL-12 antibody. In this assay, the sample, the labeledrhIL-12 standards, and the anti-hIL-12 antibody are combined and theamount of labeled rhIL-12 standard bound to the unlabeled antibody isdetermined. The amount of hIL-12 in the sample is inversely proportionalto the amount of labeled rhIL-12 standard bound to the anti-hIL-12antibody.

The antibodies and antibody portions of the invention are capable ofneutralizing IL-12 activity in vitro and in vivo, in one aspect, ahIL-12 activity. Accordingly, the antibodies and antibody portions ofthe invention can be used to inhibit IL-12 activity, e.g., in a cellculture containing IL-12, in human subjects or in other mammaliansubjects having IL-12 with which an antibody of the inventioncross-reacts (e.g., primates such as baboon, cynomolgus and rhesus). Ina one aspect, the invention provides an isolated human antibody, orantigen-binding portion thereof, that neutralizes the activity of humanIL-12, and at least one additional primate IL-12 selected from the groupconsisting of baboon IL-12, marmoset IL-12, chimpanzee IL-12, cynomolgusIL-12 and rhesus IL-12, but which does not neutralize the activity ofthe mouse IL-12. In one aspect, the IL-12 is human IL-12. For example,in a cell culture containing, or suspected of containing hIL-12, anantibody or antibody portion of the invention can be added to theculture medium to inhibit hIL-12 activity in the culture.

In another aspect, the invention provides a method for inhibiting IL-12activity in a subject suffering from a disorder in which IL-12 activityis detrimental. IL-12 has been implicated in the pathophysiology of awide variety of disorders (Windhagen et al., (1995) J. Exp. Med. 182:1985-1996; Morita et al. (1998) Arthritis and Rheumatism. 41: 306-314;Bucht et al., (1996) Clin. Exp. Immunol. 103: 347-367; Fais et al.(1994) J. Interferon Res. 14:235-238; Pyrronchi et al., (1997) Am. J.Path. 150:823-832; Monteleone et al., (1997) Gastroenterology.112:1169-1178, and Berrebi et al., (1998) Am. J. Path 152:667-672;Pyrronchi et al. (1997) Am. J. Path. 150:823-832, the entire teachingsof which are incorporated herein by reference). The invention providesmethods for inhibiting IL-12 activity in a subject suffering from such adisorder, which method comprises administering to the subject anantibody or antibody portion of the invention such that IL-12 activityin the subject is inhibited. In one aspect, the IL-12 is human IL-12 andthe subject is a human subject. Alternatively, the subject can be amammal expressing IL-12 with which an antibody of the inventioncross-reacts. Still further the subject can be a mammal into which hasbeen introduced hIL-12 (e.g., by administration of hIL-12 or byexpression of an hIL-12 transgene). An antibody of the invention can beadministered to a human subject for therapeutic purposes. Moreover, anantibody of the invention can be administered to a non-human mammalexpressing a IL-12 with which the antibody cross-reacts for veterinarypurposes or as an animal model of human disease. Regarding the latter,such animal models may be useful for evaluating the therapeutic efficacyof antibodies of the invention (e.g., testing of dosages and timecourses of administration).

As used herein, the phrase “a disorder in which IL-12 activity isdetrimental” is intended to include diseases and other disorders inwhich the presence of IL-12 in a subject suffering from the disorder hasbeen shown to be or is suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes to aworsening of the disorder. Accordingly, a disorder in which IL-12activity is detrimental is a disorder in which inhibition of IL-12activity is expected to alleviate the symptoms and/or progression of thedisorder. Such disorders may be evidenced, e.g., by an increase in theconcentration of IL-12 in a biological fluid of a subject suffering fromthe disorder (e.g., an increase in the concentration of IL-12 in serum,plasma, synovial fluid, etc. of the subject), which can be detected,e.g., using an anti-IL-12 antibody as described above. There arenumerous examples of disorders in which IL-12 activity is detrimental.In one aspect, the antibodies or antigen binding portions thereof, canbe used in therapy to treat the diseases or disorders described herein.In another aspect, the antibodies or antigen binding portions thereof,can be used for the manufacture of a medicine for treating the diseasesor disorders described herein. The use of the antibodies and antibodyportions of the invention in the treatment of a few non-limitingspecific disorders is discussed further below.

Interleukin 12 plays a critical role in the pathology associated with avariety of diseases involving immune and inflammatory elements. Thesediseases include, but are not limited to, rheumatoid arthritis,osteoarthritis, juvenile chronic arthritis, Lyme arthritis, psoriaticarthritis, reactive arthritis, spondyloarthropathy, systemic lupuserythematosus, Crohn's disease, ulcerative colitis, inflammatory boweldisease, insulin dependent diabetes mellitus, thyroiditis, asthma,allergic diseases, psoriasis, dermatitis scleroderma, atopic dermatitis,graft versus host disease, organ transplant rejection, acute or chronicimmune disease associated with organ transplantation, sarcoidosis,atherosclerosis, disseminated intravascular coagulation, Kawasaki'sdisease, Grave's disease, nephrotic syndrome, chronic fatigue syndrome,Wegener's granulomatosis, Henoch-Schoenlein purpurea, microscopicvasculitis of the kidneys, chronic active hepatitis, uveitis, septicshock, toxic shock syndrome, sepsis syndrome, cachexia, infectiousdiseases, parasitic diseases, acquired immunodeficiency syndrome, acutetransverse myelitis, Huntington's chorea, Parkinson's disease,Alzheimer's disease, stroke, primary biliary cirrhosis, hemolyticanemia, malignancies, heart failure, myocardial infarction, Addison'sdisease, sporadic, polyglandular deficiency type I and polyglandulardeficiency type II, Schmidt's syndrome, adult (acute) respiratorydistress syndrome, alopecia, alopecia greata, seronegative arthopathy,arthropathy, Reiter's disease, psoriatic arthropathy, ulcerative coliticarthropathy, enteropathic synovitis, chlamydia, yersinia and salmonellaassociated arthropathy, spondyloarthopathy, atheromatousdisease/arteriosclerosis, atopic allergy, autoimmune bullous disease,pemphigus vulgaris, pemphigus foliaceus, pemphigoid, linear IgA disease,autoimmune haemolytic anemia, Coombs positive haemolytic anaemia,acquired pernicious anemia, juvenile pernicious anaemia, myalgicencephalitis/Royal Free Disease, chronic mucocutaneous candidiasis,giant cell arteritis, primary sclerosing hepatitis, cryptogenicautoimmune hepatitis, Acquired Immunodeficiency Disease Syndrome,Acquired Immunodeficiency Related Diseases, Hepatitis C, common variedimmunodeficiency (common variable hypogammaglobulinaemia), dilatedcardiomyopathy, female infertility, ovarian failure, premature ovarianfailure, fibrotic lung disease, cryptogenic fibrosing alveolitis,post-inflammatory interstitial lung disease, interstitial pneumonitis,connective tissue disease associated interstitial lung disease, mixedconnective tissue disease associated lung disease, systemic sclerosisassociated interstitial lung disease, rheumatoid arthritis associatedinterstitial lung disease, systemic lupus erythematosus associated lungdisease, dermatomyositis/polymyositis associated lung disease,Sjodgren's disease associated lung disease, ankylosing spondylitisassociated lung disease, vasculitic diffuse lung disease, haemosiderosisassociated lung disease, drug-induced interstitial lung disease,radiation fibrosis, bronchiolitis obliterans, chronic eosinophilicpneumonia, lymphocytic infiltrative lung disease, postinfectiousinterstitial lung disease, gouty arthritis, autoimmune hepatitis, type-1autoimmune hepatitis (classical autoimmune or lupoid hepatitis), type-2autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune mediatedhypoglycemia, type B insulin resistance with acanthosis nigricans,hypoparathyroidism, acute immune disease associated with organtransplantation, chronic immune disease associated with organtransplantation, osteoarthrosis, primary sclerosing cholangitis,idiopathic leucopenia, autoimmune neutropenia, renal disease NOS,glomerulonephritides, microscopic vasulitis of the kidneys, lymedisease, discoid lupus erythematosus, male infertility idiopathic orNOS, sperm autoimmunity, multiple sclerosis (all subtypes),insulin-dependent diabetes mellitus, sympathetic ophthalmia, pulmonaryhypertension secondary to connective tissue disease, Goodpasture'ssyndrome, pulmonary manifestation of polyarteritis nodosa, acuterheumatic fever, rheumatoid spondylitis, Still's disease, systemicsclerosis, Takayasu's disease/arteritis, autoimmune thrombocytopenia,idiopathic thrombocytopenia, autoimmune thyroid disease,hyperthyroidism, goitrous autoimmune hypothyroidism (Hashimoto'sdisease), atrophic autoimmune hypothyroidism, primary myxoedema,phacogenic uveitis, primary vasculitis and vitiligo. The humanantibodies, and antibody portions of the invention can be used to treatautoimmune diseases, in particular those associated with inflammation,including, rheumatoid spondylitis, allergy, autoimmune diabetes, andautoimmune uveitis.

In certain aspects, the antibodies of the invention or antigen-bindingportions thereof, are used to treat rheumatoid arthritis, Crohn'sdisease, multiple sclerosis, insulin dependent diabetes mellitus andpsoriasis.

8.2 Use of Anti-IL-12 Antibody in Rheumatoid Arthritis

Interleukin-12 has been implicated in playing a role in inflammatorydiseases such as rheumatoid arthritis. Inducible IL-12p40 message hasbeen detected in synovia from rheumatoid arthritis patients and IL-12has been shown to be present in the synovial fluids from patients withrheumatoid arthritis (see, e.g., Morita et al., (1998) Arthritis andRheumatism 41: 306-314, the entire teaching of which is incorporatedherein by reference). IL-12 positive cells have been found to be presentin the sublining layer of the rheumatoid arthritis synovium. The humanantibodies, and antibody portions of the invention can be used to treat,e.g., rheumatoid arthritis, juvenile rheumatoid arthritis, Lymearthritis, rheumatoid spondylitis, osteoarthritis and gouty arthritis.Typically, the antibody, or antibody portion, is administeredsystemically, although for certain disorders, local administration ofthe antibody or antibody portion may be beneficial. An antibody, orantibody portion, of the invention also can be administered with one ormore additional therapeutic agents useful in the treatment of autoimmunediseases.

In the collagen induced arthritis (CIA) murine model for rheumatoidarthritis, treatment of mice with an anti-IL-12 mAb (rat anti-mouseIL-12 monoclonal antibody, C17.15) prior to arthritis profoundlysuppressed the onset, and reduced the incidence and severity of disease.Treatment with the anti-IL-12 mAb early after onset of arthritis reducedseverity, but later treatment of the mice with the anti-IL-12 mAb afterthe onset of disease had minimal effect on disease severity.

8.3 Use of Anti-IL-12 Antibody in Crohn's Disease

Interleukin-12 also plays a role in the inflammatory bowel disease,Crohn's disease. Increased expression of IFN-γ and IL-12 occurs in theintestinal mucosa of patients with Crohn's disease (see, e.g., Fais etal., (1994) J. Interferon Res. 14: 235-238; Pyrronchi et al., (1997)Amer. J. Pathol. 150: 823-832; Monteleone et al., (1997)Gastroenterology 112: 1169-1178; Berrebi et al., (1998) Amer. J. Pathol.152: 667-672, the entire teachings of which are incorporated herein byreference). Anti-IL-12 antibodies have been shown to suppress disease inmouse models of colitis, e.g., TNBS induced colitis IL-2 knockout mice,and recently in IL-10 knock-out mice. Accordingly, the antibodies, andantibody portions, of the invention, can be used in the treatment ofinflammatory bowel diseases.

8.4 Use of Anti-IL-12 Antibody in Multiple Sclerosis

Interleukin-12 has been implicated as a key mediator of multiplesclerosis. Expression of the inducible IL-12 p40 message or IL-12 itselfcan be demonstrated in lesions of patients with multiple sclerosis(Windhagen et al., (1995) J. Exp. Med. 182: 1985-1996, Drulovic et al.,(1997) J. Neurol. Sci. 147:145-150, the entire teachings of which areincorporated herein by reference). Chronic progressive patients withmultiple sclerosis have elevated circulating levels of IL-12.Investigations with T-cells and antigen presenting cells (APCs) frompatients with multiple sclerosis revealed a self-perpetuating series ofimmune interactions as the basis of progressive multiple sclerosisleading to a Th1-type immune response. Increased secretion of IFN-γ fromthe T cells led to increased IL-12 production by APCs, which perpetuatedthe cycle leading to a chronic state of a Th1-type immune activation anddisease (Balashov et al., (1997) Proc. Natl. Acad. Sci. 94: 599-603, theentire teaching of which is incorporated herein by reference). The roleof IL-12 in multiple sclerosis has been investigated using mouse and ratexperimental allergic encephalomyelitis (EAE) models of multiplesclerosis. In a relapsing-remitting EAE model of multiple sclerosis inmice, pretreatment with anti-IL-12 mAb delayed paralysis and reducedclinical scores. Treatment with anti-IL-12 mAb at the peak of paralysisor during the subsequent remission period reduced clinical scores.Accordingly, the antibodies or antigen binding portions thereof of theinvention nay serve to alleviate symptoms associated with multiplesclerosis in humans.

8.5 Use of Anti-IL-12 Antibody in Insulin-Dependent Diabetes Mellitus

Interleukin-12 has been implicated as an important mediator ofinsulin-dependent diabetes mellitus (IDDM). IDDM was induced in NOD miceby administration of IL-12, and anti-IL-12 antibodies were protective inan adoptive transfer model of IDDM. Early onset IDDM patients oftenexperience a so-called “honeymoon period” during which some residualislet cell function is maintained. These residual islet cells produceinsulin and regulate blood glucose levels better than administeredinsulin. Treatment of these early onset patients with an anti-IL-12antibody may prevent further destruction of islet cells, therebymaintaining an endogenous source of insulin.

8.6 Use of Anti-IL-12 Antibody in Psoriasis

Interleukin-12 has been implicated as a key mediator in psoriasis.Psoriasis involves acute and chronic skin lesions that are associatedwith a TH 1-type cytokine expression profile. (Hamid et al. (1996) J.Allergy Clin. Immunol. 1:225-231; Turka et al. (1995) Mol. Med.1:690-699, the entire teachings of which are incorporated herein byreference). IL-12 p35 and p40 mRNAs were detected in diseased human skinsamples. Accordingly, the antibodies or antigen binding portions thereofof the invention may serve to alleviate chronic skin disorders suchpsoriasis.

8.7 Uses of Anti-IL-18 Antibody Generally

Given their ability to bind to IL-18, the anti-IL-18 antibodies, orportions thereof, of the invention can be used to detect IL-18, in oneaspect, hIL-18 (e.g., in a sample matrix, in one aspect, a biologicalsample, such as serum or plasma), using a conventional immunoassay, suchas an enzyme linked immunosorbent assays (ELISA), an radioimmunoassay(RIA) or tissue immunohistochemistry. The invention provides a methodfor detecting IL-18 in a biological sample comprising contacting asample with an antibody, or antibody portion, of the invention anddetecting either the antibody (or antibody portion) bound to IL-18 orunbound antibody (or antibody portion), to thereby detect IL-18 in thesample. The antibody is directly or indirectly labeled with a detectablesubstance to facilitate detection of the bound or unbound antibody.Suitable detectable substances include various enzymes, prostheticgroups, fluorescent materials, luminescent materials and radioactivematerials. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examplesof suitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; and examples ofsuitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S, or ³H. Detectionof IL-18 in a sample may be useful in a diagnostic context, for examplein the diagnosis of a condition associated with increased IL-18, and/ormay be useful in identifying a subject who may benefit from treatmentwith an anti-IL-18 antibody.

Alternative to labeling the antibody, IL-18 can be assayed in a sampleby a competition immunoassay utilizing, e.g., rhIL-18 standards labeledwith a detectable substance and an unlabeled anti-IL-18 antibody, suchas an anti-hIL-18 antibody. In this assay, the sample, the labeledrhIL-18 standards, and the anti-hIL-18 antibody are combined and theamount of labeled rhIL-18 standard bound to the unlabeled antibody isdetermined. The amount of hIL-18 in the sample is inversely proportionalto the amount of labeled rhIL-18 standard bound to the anti-hIL-18antibody.

The antibodies and antibody portions of the invention are capable ofneutralizing IL-18 activity in vitro and in vivo, in one aspect, ahIL-18 activity. Accordingly, the antibodies and antibody portions ofthe invention can be used to inhibit IL-18 activity, e.g., in a cellculture containing IL-18, in human subjects or in other mammaliansubjects having IL-18 with which an antibody of the inventioncross-reacts (e.g., primates such as baboon, cynomolgus and rhesus). Ina one aspect, the invention provides an isolated human antibody, orantigen-binding portion thereof, that neutralizes the activity of humanIL-18, and at least one additional primate IL-18 selected from the groupconsisting of baboon IL-18, marmoset IL-18, chimpanzee IL-18, cynomolgusIL-18 and rhesus IL-18, but which does not neutralize the activity ofthe mouse IL-18. In one aspect, the IL-18 is human IL-18. For example,in a cell culture containing, or suspected of containing hIL-18, anantibody or antibody portion of the invention can be added to theculture medium to inhibit hIL-18 activity in the culture.

In another aspect, the invention provides a method for inhibiting IL-18activity in a subject suffering from a disorder in which IL-18 activityis detrimental. Interleukin 18 plays a critical role in the pathologyassociated with a variety of diseases involving immune and inflammatoryelements.

As used herein, the phrase “a disorder in which IL-18 activity isdetrimental” is intended to include diseases and other disorders inwhich the presence of IL-18 in a subject suffering from the disorder hasbeen shown to be or is suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes to aworsening of the disorder. Accordingly, a disorder in which IL-18activity is detrimental is a disorder in which inhibition of IL-18activity is expected to alleviate the symptoms and/or progression of thedisorder. Such disorders may be evidenced, e.g., by an increase in theconcentration of IL-18 in a biological fluid of a subject suffering fromthe disorder (e.g., an increase in the concentration of IL-18 in serum,plasma, synovial fluid, etc. of the subject), which can be detected,e.g., using an anti-IL-18 antibody as described above. There arenumerous examples of disorders in which IL-18 activity is detrimental.In one aspect, the antibodies or antigen binding portions thereof, canbe used in therapy to treat the diseases or disorders described herein.In another aspect, the antibodies or antigen binding portions thereof,can be used for the manufacture of a medicine for treating the diseasesor disorders described herein. The use of the antibodies and antibodyportions of the invention in the treatment of a few non-limitingspecific disorders is discussed further below.

The invention provides pharmaceutical compositions for the treatment ofdiseases or conditions which require modulation of IL-18 activity. Thesediseases or conditions include autoimmune diseases, type I diabetes,rheumatoid arthritis, graft rejections, inflammatory bowel disease,sepsis, multiple sclerosis, ischemic heart diseases (including heartattacks), ischemic brain injury, chronic hepatitis, psoriasis, chronicpancreatitis, acute pancreatitis and the like.

Accordingly, anti-IL-18 antibodies or antigen-binding portions thereof,or vectors expressing same in vivo are indicated for the treatment ofautoimmune diseases, Type I diabetes, rheumatoid arthritis, graftrejections, inflammatory bowel disease, sepsis, multiple sclerosis,ischemic heart disease including acute heart attacks, ischemic braininjury, chronic hepatitis, psoriasis, chronic pancreatitis and acutepancreatitis and similar diseases, in which there is an aberrantexpression of IL-18, leading to an excess of IL-18 or in cases ofcomplications due to exogenously administered IL-18.

8.8 Use Anti-IL-18 Antibody in Liver Injury

One aspect of the present invention is to provide for a novel means fortreating and/or preventing liver injury. It has been found that an IL-18inhibitor is effective in the prevention and treatment of liver damages.The invention therefore also relates to the use of an IL-18 inhibitorfor the manufacture of a medicament for treatment and/or prevention ofliver injury. More specifically, the invention relates to the treatmentand/or prevention of liver injuries caused by alcoholic hepatitis, viralhepatitis, immune hepatitis, fulminant hepatitis, liver cirrhosis, andprimary biliary cirrhosis.

8.9 Use Anti-IL-18 Antibody in Arthritis

It has also been found in accordance with the present invention that aninhibitor of IL-18 is effective in the therapy of arthritis. Thetherapeutic effect includes decreasing the severity of the disease, aswell as preventing the spreading of the disease. The invention thereforerelates to the use of an inhibitor of IL-18 for treatment and/orprevention of arthritis. This finding is unexpected, since from thestate of the art outlined above, it could not have been concluded that ablockade of one specific factor involved in arthritis, namelyinterleukin IL-18, would lead to the alleviation of arthritis or eventhe curing of a diseased arthritic joint.

The term “arthritis” includes all different types of arthritis andarthritic conditions, both acute and chronic arthritis, as defined forexample in the Homepage of the Department of Orthopaedics of theUniversity of Washington on Arthritis. Examples for arthritic conditionsare ankylosing spondylitis, back pain, carpal deposition syndrome,Ehlers-Danlos-Syndrome, gout, juvenile arthritis, lupus erythematosus,myositis, osteogenesis imperfecta, osteoporosis, polyartheritis,polymyositis, psoriatic arthritis, Reiter's syndrome, scleroderma,arthritis with bowel disease, Behcets's disease, children's arthritis,degenerative joint disease, fibromyalgia, infectious arthritis, Lymedisease, Marfan syndrome, osteoarthritis, osteonecrosis, Pagets Disease,Polymyalgia rheumatica, pseudogout, reflex sympathetic dystrophy,rheumatoid arthritis, rheumatism, Sjogren's syndrome, familialadenomatous polyposis and the like.

Rheumatoid arthritis (RA) causes inflammation in the lining of thejoints (the synovial membrane, a one cell layer epithelium) and/orinternal organs. The disease tends to persist for many years, typicallyaffects many different joints throughout the body and ultimately cancause damage to cartilage, bone, tendons, and ligaments. The joints thatmay be affected by RA are the joints located in the neck, shoulders,elbows, hips, wrists, hands, knees, ankles and feet, for example. Inmany cases, the joints are inflamed in a symmetrical pattern in RA.

RA is prevalent in about 1% of the population in the United States,being distributed within all ethnic groups and ages. It occurs all overthe world, and women outnumber men by 3 to 1 among those having RA.

It has been found that the administration of an IL-18 inhibitorsignificantly diminishes cartilage erosion in a murine model ofarthritis. The present invention thus also relates to the use of aninhibitor of IL-18 in the manufacture of a medicament for treatmentand/or prevention of cartilage destruction.

8.10 Use of anti-TNFα Antibody Generally

Tumor necrosis factor-α is a multifunctional pro-inflammatory cytokinesecreted predominantly by monocytes/macrophages that has effects onlipid metabolism, coagulation, insulin resistance, and endothelialfunction. TNFα is a soluble homotrimer of 17 kD protein subunits. Amembrane-bound 26 kD precursor form of TNFα also exists. It is found insynovial cells and macrophages in tissues. Cells other than monocytes ormacrophages also produce TNFα. For example, human non-monocytic tumorcell lines produce TNFα as well as CD4⁺ and CD8⁺ peripheral blood Tlymphocytes and some cultured T and B cell lines produce TNFα. It isinvolved in, but not unique to, rheumatoid arthritis, and occurs in manyinflammatory diseases. Receptors for TNFα are on several mononuclearcells, in the synovial membrane, as well as the peripheral blood andsynovial fluid. TNFα is a critical inflammatory mediator in rheumatoidarthritis, and may therefore be a useful target for specificimmunotherapy.

TNFα causes pro-inflammatory actions which result in tissue injury, suchas degradation of cartilage and bone, induction of adhesion molecules,inducing pro-coagulant activity on vascular endothelial cells,increasing the adherence of neutrophils and lymphocytes, and stimulatingthe release of platelet activating factor from macrophages, neutrophilsand vascular endothelial cells. Recent evidence associates TNFα withinfections, immune disorders, neoplastic pathologies, autoimmunepathologies and graft-versus-host pathologies.

TNFα is believed to play a central role in gram-negative sepsis andendotoxic shock, including fever, malaise, anorexia, and cachexia.Endotoxin strongly activates monocyte/macrophage production andsecretion of TNFα and other cytokines. TNFα and other monocyte-derivedcytokines mediate the metabolic and neurohormonal responses toendotoxin. Endotoxin administration to human volunteers produces acuteillness with flu-like symptoms including fever, tachycardia, increasedmetabolic rate and stress hormone release. Circulating TNFα increases inpatients suffering from gram-negative sepsis.

Thus, TNFα has been implicated in inflammatory diseases, autoimmunediseases, viral, bacterial and parasitic infections, malignancies,and/or neurodegenerative diseases and is a useful target for specificbiological therapy in diseases, such as rheumatoid arthritis and Crohn'sdisease. Beneficial effects in open-label trials with a chimericmonoclonal antibody to TNFα have been reported with suppression ofinflammation and with successful re-treatment after relapse inrheumatoid arthritis and in Crohn's disease.

Neutralizing antisera or mAbs to TNFα have been shown in mammals toabrogate adverse physiological changes and prevent death after lethalchallenge in experimental endotoxemia and bacteremia. Adalimumab (alsoknown by its trademark HUMIRA® available from Abbott Laboratories) is arecombinant human monoclonal antibody specific for TNFα. This monoclonalantibody binds to TNFα and blocks its interaction with the p55 and p75cell-surface TNFα receptors. This monoclonal antibody is quite specificfor TNFα as it appears not to inhibit the activity of TNFβ. In thepresence of complement, adalimumab lyses the surface of cells expressingTNFα.

EXAMPLES 1. The Isolation and Purification of Anti-IL-12 Antibody

1.1 Purification Strategy

Upon completion of a production cell culture operation designed toproduce IL-12 antibodies, the broth was slowly adjusted to andmaintained at pH 3.5 for a period of 1 hour, then readjusted to pH 5,followed by centrifugation (11,000×g) to remove cells and precipitatedimpurities. The centrifugation supernatant was further clarified by atwo step filtration train (Cuno 90ZA depth filtration, followed by0.45/0.2 μm filtration). Following clarification, the antibody wascaptured and further purified in a five step procedure.

The clarified acidified cell culture harvest was titrated to pH 7 with 3N NaOH in the course of 30 minutes and filtered prior to chromatography.

A 1.0 cm diameter×21.6 cm long column (17 mL bed volume) was used forthis operation at the bench scale. A 1.0 cm×15.1 cm column (11.9 mL bedvolume) was used at the early stage to generate material for the finepurification development. A 20 cm×21 cm column (6.6 L bed volume) wasused at the 300 L scale. The columns were packed with MabSelect™ resin(GE Healthcare, cat# 17-5199-04, lot# 302070); the asymmetry and Heightof an Equivalent Theoretical Plate (HETP) were measured to determine thequality of the packing.

The column was pre-equilibrated with 25 mM Tris, 100 mM NaCl, pH 7.2buffer. Following equilibration, the column was loaded with clarifiedcell culture harvest (v=300 cm/hr for the initial 35 g Ab/L resin load,then 150 cm/hr till the end of the load, 40 g/L maximum). The column wasthen washed with 3 column volumes (CV) of equilibration buffer (v=300cm/hr) followed by 3 CVs of 20 mM citric acid/NaCltrate, 0.5 M NaCl, pH6 buffer wash, followed by an additional 3 CVs of equilibration buffer.The product was then eluted with 0.1 M acetic acid, 100 mM NaCl, pH 3.5(v=300 cm/hr). The column eluate was collected from an initial A₂₈₀deflection of 0.1 AU to a reading of 0.1 AU at the trailing edge of theelution peak (2 mm flow cell path) and titrated to pH 5 immediately with1.0 M Tris, pH 10 buffer.

The dynamic binding capacity (DBC) of the MabSelect™ column wasdetermined either by a single flow rate load or dual-flow rate loadstrategy. The single flow rate load was evaluated at 300 cm/hr throughthe entire loading. The dual-flow rate load strategy was carried byloading the column up to 35 mg protein/mL resin at a linear velocity of300 cm/hr, and then reducing the linear velocity by half to allow longerresidence time for the last portion of the load. In each experiment thecolumn was overloaded to ensure the saturation of the column at thegiven loading conditions. The flow-through material for each run wasfractionated to determine the total amount of antibody loaded before 5%was detected in the break though. Titers of flow through (FT) fractionswere obtained by the Poros A™ HPLC assay.

Recovery was calculated by UV spectrophotometry at 280 nm (extinctioncoefficient of 1.42 for a 1 mg/mL solution using a 1 cm path length) forelution samples. Titers of the load were determined by the Poros A™ HPLCassay. SEC-HPLC, HCP ELISA and Protein A ELISA were carried out forpurity assessment, HCP removal and leached Protein A levels.

The MabSelect™ eluate was titrated to pH 8±0.1 with 1 M Tris buffer, pH10, and then diluted to 6.5±0.5 mS/cm with water. The diluted materialwas mixed for a period of one hour, followed by a Cuno™ delipid and0.45/0.22 μm filtration.

A 1.0 cm diameter×29 cm long column (22.8 mL bed volume) was used forthe Q Sepharose™ Fast Flow chromatography at the bench scale. A 20cm×29.5 cm column (9.27 L bed volume) was used at the 300 L scale. Thecolumns were packed with Q Sepharose™ FF resin (GE Healthcare,Piscataway, N.J., cat# 17-0510-04, lot# 296307 for the 300 L scale, lot#303189 for the bench scale); asymmetry and HETP were measured todetermine the quality of the packing.

Equilibration of the resin was accomplished with 25 mM Tris, 50 mM NaCl,pH 8. The chromatography was operated in the flow-through mode whereprocess-related impurities adsorbed to the resin and antibodies flowedthrough the column. Loading of the column was performed at 150 cm/hr,and the column flow-through (FTW) collection was started when the A₂₈₀rose above 0.2 AU. Samples of the FTW material were taken at intervalscorresponding to 30, 50, 80, 90 and 100 g of protein/L resin loadings.The column was then washed with equilibration buffer and the FTWcollection was continued until the A₂₈₀ returned to an OD of 0.2 AU. TheQ Sepharose™ FTW was diluted with an equal volume of 1.7 M (NH₄)₂SO₄, 50mM Na phosphate, pH 7 (SR-343) immediately, to prepare for loading ontothe Phenyl SepharosTMe HP column. This solution was 0.2 μfiltered andlabeled Phenyl Sepharose™ HP load.

Recovery was calculated by UV spectrophotometry at 280 nm (extinctioncoefficient of 1.42 for a 1 mg/mL solution using a 1 cm path length).SEC-HPLC, HCP ELISA and Protein A ELISA were carried out for purityassessment, HCP removal and leached Protein A removal.

A 1.0 cm diameter×15.6 cm long column (12.3 mL bed volume) was used forPhenyl Sepharose™ HP Chromatography at the bench scale. A 20 cm×15.5 cmcolumn (4.87 L bed volume) was used at the 300 L scale. The columns werepacked with Phenyl Sepharose™ HP resin (GE Healthcare, Piscatway, N.J.,cat# 17-1082-04, lot# 298138); asymmetry and HETP were measured todetermine the quality of the packing.

Equilibration of the resin was accomplished with 25 mM sodium phosphate,0.85 M ammonium sulfate, pH 7. The maximum protein loading for this stepwas ≦40 g protein per liter of resin (v=75 cm/hr). Following loading,the column was washed with 3 column volumes of 20 mM sodium phosphate,1.1 M (NH₄)₂SO₄, pH 7 (SR-290). The product was eluted by performing astep gradient using 15 mM sodium phosphate, pH 7, 0.5 M ammonium sulfateat a linear velocity of 37.5 cm/hr. The column eluate was collected froman initial A₂₈₀ deflection of 1.0 AU to a reading of 0.2 AU at thetrailing edge of the elution peak. Aggregates and other impuritiesremain bound to the resin and were stripped off the HIC column bywashing with water or 0.5 M NaOH.

Recovery was calculated by UV spectrophotometry at 280 nm (extinctioncoefficient of 1.42 for a 1 mg/mL solution using a 1 cm path length).SEC-HPLC, HCP ELISA and Protein A ELISA were carried for purityassessment, HCP removal and leached Protein A removal.

Viral filtration with the Ultipor DV20™ filter was not evaluated ateither the bench scale or the scale-up lab due to the size limitation atboth scales. Instead, an Ultipor DV50™ filter was used at the 300 Lscale to generate the pre-clinical material. At the bench scale, theproduct that eluted from the Phenyl Sepharose™ HP chromatography wasused directly for the final ultrafiltration and diafiltration operation.

A Millipore 30 kD molecular weight cut-off (MWCO) regenerated celluloseultrafilter was used for this step (UF/DF). Prior to operation, the UFsystem was sanitized with 1 M NaOH and rinsed with water.

The DV50™ filtrate was initially concentrated to 30 mg/mL protein.Continuous diafiltration with 2 volumes (DV) of 5 mM histidine, 5 mMmethionine, 0.5% sucrose, 2% (w/v) mannitol, pH 5.9 (diafiltrationbuffer) (SR-265) was performed. The retentate was further concentratedto 40 g/L, then diafiltrated with an additional 6 DVs of diafiltrationbuffer. The retentate was further concentrated to ±75 g/L. The UF systemwas then drained of product and rinsed with diafiltration buffer torecover product held up in the system. The concentrate and wash werecombined to produce the diafiltered antibody product at ±65 g/L. Thisproduct was 0.2 μm filtered and ready for final bottling.

1.2 Analysis of Sample Purity

SEC-HPLC. The purity of the antibody product was assessed bysize-exclusion chromatography SEC-HPLC.

Poros A HPLC Assay. The antibody titers of the harvest, MabSelect™ loadand flow through samples were determined by a Poros A™ quantitationcapture assay.

HCP ELISA. Host cell protein levels were evaluated using Host CellProtein—ELISA. HCP was sandwiched between two specific antibodies in anEnzyme Linked Immunosorbant Assay (ELISA).

The plate was coated with a 100 μL/well of a mixture of goat anti CHOaffinity purified antibodies (6 μg/mL each in 50 mM sodium bicarbonate,pH 9.4) for 1 hour at 37° C. while shaking at 80 rpm; the contents ofthe plate were then poured out and non-specific sites were blocked with300 μL/well of casein for 1 hour at 37° C. while shaking at 80 rpm.After blocking, the plate was washed with wash buffer (1×PBS, 0.1%Triton X-100, pH 9). Samples for the standard curve were prepared fromserially diluting a 5.2 mg/mL aliquot of CHO host cell proteins. Thediluent for all reagents was casein, heated to 37° C. and sonicated for2 minutes, unless otherwise stated. Concentrations for the standardcurve were 300, 200, 133, 89, 59, 40, 26, and 18 ng/mL. Antibodystandard was diluted 5× and used as the assay control. For the assay tobe accepted, the calculated value for the control must be in the rangeof 45-76 ng HCP/mL. Samples were diluted so that the calculated valueswould fall within the range of the standard curve. Standards, control,and samples (100 μL/well) were loaded onto the plate in duplicate. Theplate was incubated at 37° C. while shaking at 80 rpm for 1 hour. Aftersample incubation, the plate was washed with buffer and loaded with 100μL/well of biotinylated goat anti CHO antibody diluted to 3 μg/mL. Theplate was incubated at 37° C. while shaking at 80 rpm for 1 hour andthen washed. 100 μL/well of Neutravadin-Hoorse Rsadish Peroxidase (HRP)at 100 μg/mL was added and the plate incubated at 37° C. while shakingat 80 rpm for 1 hour. A final wash was performed and the plate wasdeveloped with 100 μL/well of K Blue HRP colorometric substrate for 7minutes while shaking at speed 3 on a Lab-line titer plate shaker atroom temperature. The reaction was stopped with the addition of 100μL/well of 2 M phosphoric acid and the plate was read at 450 nm. Colordevelopment is proportional to the amount of HCP present in the testsample.

Protein A ELISA. Protein A clearance was evaluated using an assay kitfrom Cygnus Technologies, Inc. (Immunoenymetric Assay for theMeasurement of Protein A in samples containing human immunoglobulin,catalog# F050H). Standards, reagents, and plate were provided in thekit. Samples were diluted to 1 mg/mL with 1×PBS and denatured at 100° C.for 5 minutes with denaturing buffer (50 μL denaturing buffer into 200μL sample). The samples were centrifuged at 6000×g for 5 minutes topellet the denatured antibody. 100 μl of biotinylated anti-Protein A and504 of supernatant from denatured samples, controls, and standards wereadded to all wells. The plate was incubated for 1 hour at roomtemperature while shaking at 180 rpm. The contents of wells wereaspirated and the plate manually washed four times with 300 μL of washsolution. Streptavidin:Alkaline Phosphatase was added to each well (100μL) and the plate was incubated at room temperature for 1 hour whileshaking at 180 rpm. After washing the plate as described above, 100 μLof Alkaline Phosphatase colorometric substrate was added to the wellsand the plate was incubated for 30 minutes at room temperature whileshaking at 180 rpm. The plate was read at 405 and 492 nm. If theabsorbance for the 16 ng/mL sample was <1.2, the plate was incubated anadditional 30 minutes and re-read. Color development is proportional tothe amount of Protein A present in the test sample.

1.3 Results of Purification Strategy

Protein A chromatography. The MabSelect™ Protein A chromatography wasused to capture anti-IL-12 antibodies and reduce product and processrelated impurities such as single and double light chain fragments, CHOhost cell proteins (HCPs) and DNA, etc. Data from both bench scale and300 L bioreactor scale showed good protein recovery and HCP clearance(Tables 1 and 2). A representative chromatogram from the 300 Lbioreactor scale was provided in FIG. 4.

TABLE 1 Summary of recovery and purity for the new process atbench-scale Leached Recovery Purity HCP Protein A Process step (%) (%monomer) (ng/mg Ab)* (ng/mg Ab) Clarification 91 ND** 211195.7 0.14MabSelect ™ 95 97.5 1121.9 9.36 Q Seph ™ FF 96 96.9 34.5 8.21 Phenyl HP90 99.2 11.1 0.65 Overall 81 ND** ND** ND** *1 Day HCP ELISA assay. SOPPD-122. **ND—not determined.

TABLE 2 Summary of the new process at 300 liter scale Leached RecoveryPurity HCP Protein A Process step (%) (% monomer) (ng/mg Ab)* (ng/mg Ab)Clarification 91 ND** 141753 ND** MabSelect ™ 95 94.44 <2117.6 7.6Delipid 97 95.97 58 3.4 filtration Q Seph ™ FF 93 96.21 8 3.7 PhenylSeph ™ 92 99.72 4 2.3 HP DV50 ™ 95 99.64 ND** ND** UF/DF 99 99.59 ND**ND** Bottling 99 99.55 5 0.3 Overall 67 NA*** NA*** NA*** *1 Day HCPELISA assay, SOP PD-122. **ND = not determined. ***NA = not applicable.

Data shown in Table 3 demonstrated that the dynamic binding capacity ofthe MabSelect™ chromatography was affected by load flow rate,specifically by residence time. When loaded at a constant linearvelocity of 300 cm/hr (3.4 minutes residence time), the DBC was 44.3 mgprotein/mL resin. When the dual-flow rate loading strategy (300 cm/hr upto 35 g/L, switching to 150 cm/hr resulted in the DBC increasing by 17%to 51.7 mg protein/mL resin. This increase of the DBC was accomplishedby doubling residence time for the latter portion of the load.Theoretically, in the initial stage of column loading, binding occurspredominately at the bead surface, sites which are easily accessible.This permits the use of faster flow rates with little impact on bindingkinetics. Once all the readily accessible sites are occupied, proteinsneed to diffuse into pores to bind to the less readily accessible sites.Kinetically this is a slower process and therefore, the flow ratesshould be reduced accordingly to maximize binding.

TABLE 3 Effect of flow rate on the DBC of the MabSelect ™ column Experi-DBC at 5% BT Load linear velocity ment # (mg Ab/mL resin) Single flow300 cm/hr Exp. 1 43.7 rate load Exp. 2 44.9 Average 44.3 Dual-flowInitial linear velocity of Exp. 3 51.6 rate load 300 cm/hr till 35 mgAb/mL Exp. 4 51.7 resin load, 2nd linear Average 51.7 velocity of 150cm/hr until appearance of antibody in the load flow through

The double light chain amount was reduced from 0.5% to 0.2% (accordingto HPLC-SEC) by implementing an intermediate wash step. Several washbuffers were evaluated including 0.5 M NaCl, 25 mM sodium acetate, 0.5 MNaCl, pH 5.0 or pH 5.5 and 20 mM citric acid/Na citrate, 0.5 M NaCl, pH6. Data from both HPLC-SEC and SDS-PAGE (FIG. 5) showed a good clearanceof low molecular weight (LMW) species from all the experiments. When thewash was performed at pH 6, the same clearance of LMW was observedcompared to the lower pH washes, while the 95% protein recovery wascomparable to runs without the intermediate wash step.

Q Sepharose™ Fast Flow chromatography. The Q Sepharose™ FF column wasused to reduce negatively charged process and product related impuritiessuch as DNA and host cell proteins. This and subsequent steps constitutethe fine purification steps in the overall process.

Prior to the chromatography, load sample preparation began with theadjustment of the pH and conductivity of the MabSelect™ eluate. The pHof the eluate was adjusted from pH 5 to pH 8 using 1 M Tris, pH 10;immediately followed by a lowering of the conductivity from ˜15 mS to 7mS with water. Once the conductivity of MabSelect™ eluate approached 7mS/cm, precipitation started to occur and was allowed to continue forone hour. The precipitated material was removed by a Cuno™ delipid or30ZA depth filter, followed by a 0.45/0.2 μm filtration. Proteinrecovery of this load preparation step was 96.3% according to the UVabsorbance at 280 nm, indicating that the majority of the precipitatedmaterial was not antibody. This was also confirmed by SDS-PAGE analysisof the precipitates (FIG. 6).

The Q Sepharose™ FF column was operated in a flow through mode. Arepresentative chromatography profile is shown in FIG. 7. No apparentbreak though of HCPs were observed when loaded at 80 g Ab/L resin at the300 L bioreactor scale (Table 4). Once the loading reached 90 g Ab/Lresin, the HCP level doubled in the flow through wash (FTW), indicatingthat the resin was reaching its binding capacity. HCP levels continuedto increase at 100 g Ab/L resin loading (Table 4). This represents asignificant improvement over the current antibody manufacturing process.Maximum loading is 50 g Ab/L resin. This loading represents a 60%increase of the binding capacity for the Q Sepharose™ operation. Theincrease in load capacity is due to the use of an affinity resin such asProtein A vs. a cation exchanger for the capture step, resulting in muchcleaner feed streams for the Q Sepharose™ unit operation.

The protein recovery is typically high for this step. Data from bothbench and the 300 L bioreactor scale runs resulted in comparable stepyields (96% and 93%) (Tables 1 and 2). HCP reduction (97% and 85%) wasalso achieved at both scales (Tables 1 and 2).

TABLE 4 Effect of load amount on HCP clearance of the QFF column Q loadamount HCP (ng/mL)* Ab (mg/mL) HCP (ng/mg) Q load depth filtered 436.3067.54 58 Q FTW 30% load** 104.122 8.66 12 Q FTW 50% load** 61.642 8.59 7Q FTW 80% load** 61.867 8.80 7 Q FTW 90% load** 132.955 8.80 15 Q FTW100% load** 157.683 8.52 17 Q FTW pool 62.808 8.24 8 *1 day HCP ELISAassay, SOP PD-122. **100% load = 100 g Ab/L resin load.

HIC chromatography. HIC chromatography was used to remove aggregated andfragmented antibody molecules from the intact antibodies based on theirdifferences in hydrophobicity. In the case of anti-IL-12 antibodies,aggregates are more hydrophobic than the monomers, hence bound tighterto the HIC column. When the column is eluted with 0.5M ammonium sulfate,monomeric Ab is eluted off, whereas aggregates remain bound to thecolumn and come off only in the water strip. In contrast, the fragmentedantibody molecules or low molecular weigh species (LMW) are lesshydrophobic than the intact antibodies and hence are not bound astightly to the HIC chromatography resin. Most of the LMW are eluted offthe column prior to the main elution peak. Since the upstream MabSelect™wash step was able to remove a majority of the LMW from the processstream; the HIC process was designed mainly to remove aggregatedantibodies, Protein A leachables, residual HCPs and other process andproduct related impurities to achieve final product specifications. Arepresentative chromatography elution profile is provided in FIG. 8.

Data from both the bench and 300 L scale showed ≧90% recovery, and goodaggregate removal (>99% purity) by HIC chromatography (see Tables 1 and2). Leached Protein A in the Q FTW material was reduced from 8.21 to0.65 ng/mg Ab (ppm) by the HIC chromatography operation (see Table 1).

In comparison to the current manufacturing process (e.g., cationexchange capture), Protein A affinity capture based process was able topurify anti-IL-12 antibodies from CHO cell culture harvest with a higheryield and better HCP removal.

2 The Isolation and Purification of anti-TNFα Antibody

2.1 The Purification Strategy

Cell Culture Harvest Clarification. Cell culture day 13 fermentationharvest broth and a clarified day 15 fermentation harvest material wereused as starting material in this example. Cell culture broth wascentrifuged at 3000 rpm for 30 minutes with Beckman Coulter Allegra™ 6RCentrifuge. The collected supernatant was filtered through Delipidfilter (CUNO ZeraPlus® BC0030A DELI—BioCap™ 30 disposable capsule) atflow rate of 20 mL/min. The clarified harvest was then aliquoted andstored at −80° C. The frozen harvest was thawed and filtered with a 0.22μm Corning® Cellulose Acetate filter before loading on to the column.The harvest broth was centrifuged with a disc-stack centrifuge andfurther filtered through depth filter and delipid filter. The filtratewas stored at 4° C. before loading on the column. The clarificationprocess was performed at room temperature.

Protein A Affinity Chromatography. A 10 mL (1.6×5 cm) column and a 40.2mL (1.6×20 cm) column were packed with MabSelect™ resin (GE Healthcare).An AKTA Explorer™ chromatography system was used for the operations.Column were packed using 0.5 M NaCl. Table 5 displays the chromatographyconditions. The elution peak fractions were combined and held for 1 hourbefore being neutralized to pH 8.0 with 3M Trolamine. The process wasperformed at room temperature.

TABLE 5 Protein A affinity Chromatography conditions Linear VolumeVelocity* Step Solution (CV) (cm/h) Rinse Water 5 400 Equilibration 1XPBS 5 400 Load See sample info variable 400 Wash 1X PBS 5 400 ElutionAcetic acid buffer w/pH 3.2 5 400 regeneration 1.0% Acetic Acid/0.5MNaCl 5 400 sanitization 6.0 M Guanidine 5 100 every cycle Rinse Milli QWater 5 400 Storage 50 mM Na Acetate pH 5, 20% 3 100 ethanol *Linearvelocity was 100 cm/h for 5 cm height column and 400 cm/h for 20 cmheight column to keep the same residence time.

Q Load Material Preparation. The neutralized eluate from two Protein Aruns were combined and diluted with approximately 0.5˜1.0 time withMilli Q water to achieve a conductivity of 4.5±5.5 mS/cm (25° C.). Thediluted sample was filtered through a depth filter (CUNO Zeta PlusBioCap® BC0030A30ZA) at a flow rate of 20 mL/min followed with a 0.45 μmcellulose acetate filter (Corning, Inc.) and then 0.22 mL/min celluloseacetate filter (Corning, Inc.). The sample was then ready to load ontothe Q column.

Q Sepharose™ Anion Exchange Chromatography. A 60.3 mL (1.6×30 m) and a40.2 mL (1.6×20 m) column were packed with Q Sepharose™ Fast Flow resin(GE Healthcare) and operated by AKTA Explorer™ chromatoragphy system.Table 6 displays the chromatography conditions. The process wasperformed at 12° C. Column flow through samples were collected forPhenyl HP chromatography purification.

TABLE 6 Q Sepharoses chromatography condition Linear Volume VelocityStep Solution (CV) (cm/h) Equilibration 25 mM Trolamine, 40 mM NaCl, 5149 pH 8.0 Load See sample info variable 149 Wash 25 mM Trolamine, 40 mMNaCl, 5 149 pH 8 Regeneration 25 mM NaPi, 1 M NaCl, pH 7.0 3 149 RinseWFI water 2 149 Sanitize 1M NaOH 3 74.6 Rinse WFI water 3 74.6 Storage25 mM NaPi, 20% iPrOH 5 74.6

Phenyl Sepharose™ HP Chromatography. A 30.2 mL (1.6×15 cm) column waspacked with Phenyl Sepharose™ HP resin (GE Healthcare) and operated byAKTA Explorer chromatography system. The Q flow through was diluted with2.2 M (NH₄)₂SO₄/40 mM NaH₂PO₄ buffer at 1:1 ratio to achieve aconductivity of 147±11 mS/cm for loading onto Phenyl Sepharose™ HPcolumn. The main elution peak was collected. Table 7 displays thechromatography conditions. The process was performed at 12° C.

TABLE 7 Phenyl HP chromatography condition Linear Volume Velocity StepSolution (CV) (cm/h) Equilibration 1.1M(NH4)SO4, 20 mM Na—PO4 5 75 LoadSee sample info variable 75 Wash 1.1M(NH4)SO4, 20 mM Na—PO4 5 75 Elution0.6M-0.65M AmSO4, 11.4 mM 3 37.5 NaPi Regeneration 25 mM Na—PO4, 20%iPrOH 3 37.5 Rinse Milli Q water 3 37.5 Sanitization 1 M NaOH 3 37.5

2.2 Analysis of Sample Purity

pH. A pH meter was calibrated using two pH standard solutions (pH 4 and7 or pH 7 and 10) immediately before use. The measured values werewithin the calibration points. A Corning Pinnacle 545 pH meter was used.

Conductivity. Conductivity was measured at 25° C., using a RadiometerAnalytical CDM210 conductivity meter.

UV spectroscopy A₂₈₀. UV A₂₈₀ was measured, using an Agilent UV8453spectrophotometer. The extinction coefficient used for adalimumab is1.39 L/g·cm.

Poros A HPLC. Antibody concentration was determined by Poros A (ProteinA) HPLC. Sample dilutions were applied to achieve readings within thecalibration range. A Shimadzu HPLC system was configured with a Poros AImmunoDetection sensor cartridge (Applied Biosystems, Foster City,Calif.). The column was maintained at ambient temperature. The systemwas run at 2 mL/minute. Autosampler tray temperature was set at 4° C.Absorbance was monitored at 280 nm. Buffer A was 1×PBS. Buffer B was 0.1M acetic acid and 150 mM sodium chloride. The sample was injected andadalimumab was eluted using 100% Buffer B.

WCX-10 HPLC. WCX-10 HPLC (weak cation exchange) analysis was performed.A Shimadzu HPLC system was configured with a Dionex ProPac WCX-10analytical column and a guard column (Dionex Corporation, Sunnyvale,Calif.). Both columns were maintained at 30° C. The system was run at 1mL/minute. Auto sampler tray temperature was set at 4° C. Absorbance wasmonitored at 280 nm. Buffer A was 10 mM sodium phosphate dibasic, pH 7.5and Buffer B was 10 mM sodium phosphate dibasic/500 mM sodium chloride,pH 5.5. The sample was injected and adalimumab was eluted using thegradient method as following.

Time (min) Buffer A (%) Buffer B (%) 0.0 94 6 20.0 84 16 22.0 0 100 26.00 100 28.0 94 6 34.0 94 6

Size exclusion Chromatography (SEC) HPLC. SEC analysis was performed.Sample dilutions were applied to achieve readings within the standardcurve. A Shimadzu HPLC system was configured with a Superose™ 6 10/300GL column (GE Healthcare). The column was maintained at ambienttemperature. The system was run at 0.5 mL/minute. Autosampler traytemperature was set at 4° C. Absorbance was monitored at 214 nm. BufferA was 20 mM sodium phosphate dibasic/150 mM sodium chloride. The samplewas injected and adalimumab was run isocratically using 100% Buffer A.

HCP ELISA. HCP ELISA was performed as outlined above. Sample dilutionswere applied to achieve readings within the calibration range.

Protein A ELISA assay. Leachable Protein A concentration analysis wasperformed as outlined above. Immunoenzymetric Assay for the Measurementof Protein A samples containing human immunoglobulin assay pre-coatedkits (Catalog #F050H) including the sample diluent (Catalog #1028) werepurchased from Cygnus Technologies, Inc. Block heater (Type 17600Dry-Bath, Barnstead/Thermolyne) was used for sample denaturing.Incubator (max 4000, Barnstead/Lab-Line) was used for sample incubation.Plate reader (Spectramax Plus, Molecular Devices) was used forabsorbance reading at 405 nm and 495 nm.

SDS-PAGE. Samples were diluted with 1×PBS buffer to a designedconcentration, then an equal volume of 2× loading buffer (Invitrogen™Novex® Tris-Glycine SDS Sample Buffer (2×), Cat# LC2676) was added toeach diluted sample. For reducing SDS-PAGE, one tenth volume of reducer(Invitrogen™ NaPAGE® Sample Reducing Agent (10×), Cat# NP0004) was addedinto each sample. 20 μL of each prepared sample was loaded in each wellon the gel (Invitrogen™ 12% Tris-Glycine Gel). The gel was run under 1×Tri-Glycine SDS running buffer (diluted from 10× Tris-Glycine-SDS bufferfrom ABC Storage Room) at constant voltage of 125 V and 35 mA for 108minutes. SimpleBlue™ SafeStain (Invitrogen™, Cat# LC6060) was used tostain the gel for 2 hours and destained with Milli Q water until clearbends appeared. SeeBlue® Plus Prestained Standard (1×) (Invitrogen™ Cat#LC5925) was used as a marker in this study. Each prepared sample wasloaded on the base of equal total amount of antibody.

2.3 Results of Purification Strategy

MabSelect™ Dynamic Binding Capacity. The dynamic binding capacity of aresin under given conditions can be estimated by over-loading the columnand analyzing the resultant breakthrough of product. Typically, thesetting for maximum binding capacity is to determine the loadingcorresponding to 10% breakthrough and then reducing this by 10 to 20% asa safety margin to assure that process measurement variability and theProtein A ligand leached with the time to be covered. The 10%breakthrough point for MabSelect™ resin is at 37.4 g antibody per L ofresin (FIG. 9). This suggested a maximum recommended resin bindingcapacity of 32 g/L resin at a flow rate of 400 cm/hr under the studiedconditions. 32 g/L load amount was used in this example.

Antibody Recovery Yield. There were two runs performed for product yieldand quality study. Harvest broth were clarified as described above.Filtrates were loaded onto the MabSelect™ column and run as describedabove. The eluates pH (pH 4.06) were higher than the sufficient viralreduction/inactivation pH (pH 3.5), therefore the eluate pool wasadjusted to pH 3.5 with 10× diluted glacial acetic acid (J. T. Baker9522-03). In the Q Sepharose™ chromatograph step, 30 g/L were loaded inRun1 and 40 g/L in Run 2 to challenge the impurity clearance capabilityof the process.

Table 8 listed the antibody recovery step yields from these 2 runs and aprocess for purification not employing Protein A (“AY-04”).

TABLE 8 Recovery yields in the studied Protein A process and AY-04process Protein A Process AY-04 Step Name Run 1 Run 2 Mapping Data *Harvest Filtrate Protein A Capture 91.81 97.12 86~91% Viral InactiveFiltration 91.00 97.65 97~98% Q Sepharose ™ CC 93.87 95.91 88~101% Phenyl Sepharose ™ CC 91.57 96.63 87~91% Overall Chromatography 71.8187.90 72-82% * Data from Tech-report ABC/TO R348

Host Cell Proteins (HCP) are the major impurities generated by CHOcells. Besides impurities carried in harvest material, Protein A mayleach from the protein A resin matrix into the product pool as a processrelated impurity. A good process must demonstrate its ability to clearHCP and leachable Protein A.

HCP Clearance. Batch harvest material was applied to a MabSelect™(2.6×20 cm) column. HCP levels in the eluate were measured as describedabove and compared to the Fractogel™ S eluate in an AY-04 process. Thedata demonstrated that MabSelect™ chromatography removed 99.9% HCPloaded and the Fractogel™ S chromatography of AY04 removed 88˜93% HCPloaded. As expected, Protein A affinity resin had better HCP clearancecapability than Fractogel™ S cation exchange resin.

Protein A Leachables. To detect Protein A leached from the resin matrix,a commercial ELISA kit (Cygnus Technologies, Inc) was used to determineProtein A content. The Protein A leached during the affinity step wasapproximately 6 ng/mg-antibody in the eluate. After fine purificationwith Q Sepharose™ and Phenyl HP, the leached protein A was significantlyreduced to approximately 0.02 ng/mg.

SDS-PAGE. A denatured reducing SDS-PAGE was conducted to compare theintermediate differences between Protein A process and AY-04 process. 2μg of antibody of each sample was loaded. No obvious difference isobserved in the gel (FIG. 10).

MabSelect™ Elution pH Study. In the case of Protein A affinity resin,antibody is bound at neutral pH and eluted under acidic condition. Sincelow pH viral reduction/inactivation is very efficient and widely used inmonoclonal antibodies purification processes, the viralreduction/inactivation step can be simplified by holding the acidicMabSelect™ eluate for a sufficient time. The effect of elution pH onantibody recovery yield and product quality were studied. Elution aroundpH 3.2 was selected as the MabSelect™ elution pH based on its highestrecovery yield and acceptable product quality in terms of monomerpercentage and total lysine variants.

FIG. 11 demonstrates that a yield of at least 90% was obtained for anelution pH range from 2.5 to 3.8 and a significant decrease in yieldoccurs at pH 4. This suggests that at an elution buffer pH higher thanpH 4 could not sufficiently elute antibody from MabSelect™ resin.

Impact on Antibody Aggregation. All of antibody (Adalimumab) monomerlevels in MabSelect™ eluates were higher than 92% (FIG. 12). They allmet the in-process action limits (SEC HPLC monomer≧92%) of Fractogel™Chromatography in AY-04 process at an elution buffer pH range of 2.5 to3.8, although higher elution pH generated less aggregates. Eluateholding time under acidic condition for 1 to 3 hours did notsignificantly affect antibody aggregation.

3. Determination of Host Cell Protein Concentration in anti-IL-12Antibody Compositions

This procedure describes the testing methodology for the determinationof residual Host Cell Protein concentration in anti-IL-12 antibodysamples. Enzyme Linked Immunosorbent Assay (ELISA) is used to sandwichthe Host Cell Protein (Antigens) between two layers of specificantibodies. This is followed by the blocking of non-specific sites withCasein. The Host Cell Proteins are then incubated during which time theantigen molecules are captured by the first antibody (Coating Antibody).A second antibody (anti-Host Cell Protein Biotinylated) is then addedwhich fixes to the antigen (Host Cell Proteins). NeutravidinHRP-conjugated is added which binds to the Biotinylated anti-Host CellProtein. This is followed by the addition of K blue substrate. Thechromogenic substrate is hydrolyzed by the bound enzyme conjugatedantibody, producing a blue color. Reaction is stopped with 2M H₃PO₄,changing color to yellow. Color intensity is directly proportional tothe amount of antigen bound in the well.

Preparation of 50 mM Sodium Bicarbonate (Coating Buffer), pH 9.4. To a 1L beaker add: 900 mL Milli-Q water; 4.20 g±0.01 g Sodium Bicarbonate.Stir until completely dissolved. Adjust pH to 9.4 with 1 N NaOH.Transfer to a 1 L volumetric flask and bring to volume with Milli-Qwater. Mix by inversion until homogeneous. Filter through a 0.22 μmsterile filter unit. Store at nominal 4° C. for up to 7 days from thedate of preparation.

Preparation of 0.104 M Na₂HPO₄*7H₂O, 1.37 M NaCl, 0.027 M KCl, 0.0176 MKH₂PO₄, pH=6.8-6.9 (10×PBS). Add approximately 400 mL of Milli-Q waterto a glass beaker. Add 13.94 g±0.01 g of Na₂HPO₄×7H₂O. Add 40.0 g±0.1 gof NaCl. Add 1.00 g 0.01 g of KCl. Add 1.20 g±0.01 g of KH₂PO₄. Stiruntil homogeneous. Transfer to a 500 mL volumetric flask. QS to 500 mLvolume with Milli-Q water. Mix by inversion. Filter through a 0.2 μmsterile filter unit. Store at room temperature for up to 7 days.

Preparation of 1×PBS+0.1% Triton X-100, pH 7.40: (Plate Wash Buffer). Ina 4 L graduated cylinder, mix 400 mL 10×PBS (step 5.2) with 3500 mLMilli-Q Water. Check pH, and adjust if necessary to 7.40±0.05 with 1 NHCl or 1 N NaOH. Bring to volume with Milli-Q water. Tightly parafilmthe cylinder and mix by inversion until homogeneous. Transfer to a 4 Lbottle. Remove 4 mL of the 1×PBS and discard. Add 4 mL of triton X-100to the 3996 mL of 1×PBS. Place on stir plate and stir to completelydissolve. Filter the amount of plate wash buffer needed for dilutionbuffer preparation through a 0.22 μm sterile filter unit. Store at roomtemperature for up to 7 days.

Preparation of Coating Antibody Mixture: goat anti CHO 599/626/748 (lot# G11201@1.534 mg/mL), affinity purified: NOTE: Stocks stored at nominal−80° C. in vials. Prepare aliquots. Take out one aliquot per plate attime of use. Immediately before use: Dilute antibody mixture to have afinal concentration of 4 μg/mL in cold 50 mM Sodium Bicarbonate asfollows. For example: add 31 μLs coating antibody mixture to 11969 μLscold coating buffer. Mix gently by inversion.

Preparation of Biotinylated goat anti Host Cell Protein Mixture,599/626/748 (lot# G11202@0.822 mg/mL): NOTE: Stocks stored at nominal−80° C. in vials. Prepare aliquots. Take out one aliquot per plate attime of use. Immediately before use: dilute biotinylated antibodymixture to have a final concentration of 1 μg/mL in 37° C.±2° C. Caseinas follows. For example: add 14.6 μLs biotinylated antibody mixture to11985 As 37° C.±2° C. Casein. Mix gently by inversion.

Preparation of Neutravidin-HRP. Reconstitute new lots (2 mg/vial) to 1mg/mL as follows: Add 400 μl of Milli-Q water to the vial, then add 1600μL 1×PBS, for a total of 2 mL. Vortex gently to mix. Store at nominal−20° C. Prepare aliquots with desired volume so that 1 aliqout per plateis used. Prepare in polypropylene tube. Qualify new lots to determineworking concentration. Assign expiry of 6 months from the date ofpreparation. For example, if the working concentration was determined tobe 0.2 μg/mL then prepare as follows. Immediately before use: thaw analiquot of Neutravidin-HRP at room temperature. Dilute the 1 mg/mLNeutravidin solution to 0.1 mg/mL (100 μg/mL) with 37° C.±2° C. Casein.For example: Dilute X10, add 50 μL of neutravidin to 450 μL of Casein.Vortex gently to mix. Further dilute the 100 μg/mL solution to 0.2 μg/mLwith 37° C.±2° C. Casein. For example: Dilute X500, add 24 μLneutravidin (100 μg/mL) to 11976 μL of Casein. Vortex gently to mix.

Preparation of 5.7 2M Phosphoric Acid (Stop Solution). Prepare a 2 MPhosphoric acid solution from concentrated phosphoric acid as follows.From the % phosphoric acid stated on the label, density (1.685 g/mL) andformula weight (98 g/mole), calculate the volume of concentratedphosphoric acid needed to prepare 500 mL of 2M phosphoric acid. Add thevolume of concentrated phosphoric acid calculated above to the flask.Bring to volume with Milli-Q water and mix by inversion untilhomogeneous. Store at ambient temperature for up to 6 months from thedate of preparation.

Preparation of Dilution Buffer (Casein diluted ×100 in 1×PBS+0.1% TritonX100, pH 7.4). Dilute 37° C.±2° C. Casein X100 in 0.22 μm sterilefiltered 1×PBS+0.1% Triton X100, pH 7.4 (from above). For example: Add 1mL of 37° C.±2° C. Casein to 99 mL 0.22 μm sterile filtered 1×PBS+0.1%Triton X100, pH 7.4. Mix well. Prepare fresh for each use.

Preparation of Standards. Host cell Protein Standards (AntigenStandards) (lot # G11203@1.218 mg/mL): NOTE: Stocks stored at nominal−80° C. in 70 μL aliquots. Thaw an aliquot at room temperature. Performserial dilutions in polypropylene tubes using Dilution buffer.

Preparation of Samples. In polypropylene tubes, dilute final bulksamples to 24 mg/mL in Dilution Buffer. Record concentration. NOTE: usethe solutions below to prepare spiked samples and to prepare the 12mg/mL solutions referenced below. In polypropylene microtubes, furtherdilute the 24 mg/mL solutions to 12 mg/mL in Dilution Buffer. Loadtriplicate wells for each of the 12 mg/mL solutions on the plate for atotal of 6 wells.

Preparation of Spike. In a polypropylene microtube, prepare a 10 ng/mLHost Cell Protein spike from the 20 ng/mL standard prepared above bydiluting it 2× with Dilution Buffer. Load three wells for the 10 ng/mLspike solution onto the plate. Use the 20 ng/mL standard solution fromstep 6.1 for spiking samples.

Preparation of Spiked Samples. In polypropylene microtubes, spike 300 μLof each 24 mg/mL final bulk solution with 300 μL of the 20 ng/mL spikesolution (6.1). Load triplicate wells for each spiked sample solutionfor a total of 6 wells.

Preparation of Control. A Control Range Must be Set for Every NewControl Stock solution, before use in routine testing. Control Stock:Prepare 150 μL aliquots of a batch of ABT-874 Drug Substance Concentrateand store frozen at nominal −80° C. for up to three years.

Preparation of Working Control. Thaw an Aliquot of Control at Roomtemperature. In polypropylene tubes, dilute control to 24 mg/mL withDilution Buffer. In polypropylene microtubes, further dilute the 24mg/mL control solution with dilution buffer to 12 mg/mL. Prepare asingle dilution and load control into 3 wells of the plate.

ELISA procedures. Fill plate wash bottle with plate wash buffer (referto step 5.3, 1×PBS+0.1% Triton X-100). Prime plate washer. Check thefollowing parameters: Parameters should be set to: Plate Type: 1 Foreach Cycle (a total of 5 cycles): Volume: 400 μls; Soak Time: 10seconds; Asp. Time: 4 seconds.

Assay Procedure. Coat plates with 100 μL/well of 4 μg/mL goat coatingantibody mixture in cold 50 mM Sodium Bicarbonate. Tap the side of theplate until the coating solution covers the bottom of the wellsuniformly, cover with sealing tape and incubate at nominal 4° C. whileshaking on plate shaker (or equivalent) at speed 3 for 18 hours±1 hour.After overnight incubation, remove plate from refrigerator and allow toequilibrate to room temperature. Shake out coating. Blot plate on papertowels. Block with 300 μL/well of 37° C.±2° C. Casein, cover withsealing tape and incubate at 37° C.±2° C. while shaking on Lab-lineEnviron plate shaker (or equivalent) at 80 rpm±5 rpm for 1 hour. Preparestandard, sample, control, spike, and spiked samples during blockingincubation. Wash the plate 5 times with Wash Buffer. Blot plate on papertowels. Using an 8-channel pipette, pipet 100 μL/well of standards,samples, spikes, spiked samples, and control into triplicate wells ofthe plate. Pipette 100 μL/well of Dilution Buffer into all empty wellsof the plate to serve as blanks. Cover with sealing tape and incubate at37° C.±2° C. while shaking on Lab-line Environ plate shaker (orequivalent) at 80 rpm±5 rpm for 1 hour. Fill out a template to use as aguide when loading plate.

Plate Reader Set-Up. Set up template, entering concentrations forstandards. Do not enter dilution factors for samples, control, spike, orspiked samples. Assign the wells containing diluent as blanks to besubtracted from all wells. Wash the plate 5 times with Wash Buffer. Blotplate on paper towels. Add 100 μL/well biotinylated goat antibody. Coverwith sealing tape and incubate at 37° C.±2° C. while shaking on Lab-lineEnviron plate shaker (or equivalent) at 80 rpm±5 rpm for 1 hour. Washthe plate 5 times with Wash Buffer. Blot plate on paper towels. Add 100μL/well Neutravidin-HRP conjugate solution. Cover with sealing tape andincubate at 37° C.±2° C. while shaking on Lab-line Environ plate shaker(or equivalent) at 80 rpm±5 rpm for 1 hour. Wash the plate 5 times withWash Buffer. Blot plate on paper towels. Add 100 μL/well cold K-Bluesubstrate, cover with sealing tape and incubate at room temperature for10 minutes (start timer as soon as substrate is added to first row),while shaking speed 3 on Lab-line titer plate shaker (or equivalent).Stop the reaction by adding 100 μL/well 2M Phosphoric Acid (Step 5.7).Place plate on a plate shaker at speed 3 for 3-5 minutes. Read plate at450 nm.

Data Analysis and Calculations. NOTE: only samples, spikes, spikedsamples, and control, with optical densities falling within thepractical quantitation limit (2.5 ng/mL standard) of the standard curveand meeting the % CV or % difference criteria stated below, areaccepted. If sample OD's fall below the 2.5 ng/mL standard, resultshould be reported as less than 2.5 ng/mL. This value should then bedivided by the diluted sample concentration (12 mg/mL) to report valuein ng/mg. If sample is high in host cell concentration causing thenon-spiked and/or the spiked sample to be above standard curve, reportvalue as >100 ng/mL. This value should then be divided by the dilutedsample concentration (12 mg/mL) to report value in ng/mg. Considersample value zero for spike recovery calculations when the sample isbelow the 2.5 ng/mL standard.

Standard Curve. Standard concentrations should be entered into theprotocol template. A quadratic curve fit is used. Coefficient ofdetermination must be=0.99 and the % CV between triplicate wells mustbe=20%. If this criteria is not met: One standard (1 level, 3 wells) maybe dropped. If the 1.25 ng/mL is dropped, only samples and spikedsamples with optical densities falling within the 2.5 ng/mL and 100ng/mL (the remaining standard curve points) optical densities areacceptable. Additionally, for the triplicates of each standard level, ifa single well is clearly contaminated or shows low binding, it may bedropped. If a well is dropped from a standard level, the remainingreplicates must have a % difference=20%. The % CV for the loweststandard, which shows OD values close to the background (blanks) of theplate, should be=30%. If one well is dropped, the % difference for theremaining replicates must be=35%. If the lowest standard is dropped,only samples and spiked samples with optical densities falling withinthe remaining standard curve level optical densities are acceptable.

Samples. % CV should be=20% between triplicate wells. Report % CVbetween triplicate wells. One well from each sample dilution may bedropped. The remaining replicates must have a % difference of =20%.Note: if non-spiked sample OD is below the 2.5 ng/mL standard OD the %difference criteria does not apply to the non-spiked results. Refer tocalculation above.

Calculate actual Host Cell Concentration in ng/mg from the mean (ng/mL)value as follows: CHO Host Cell Protein (ng/mg)=Mean “Non-spiked sampleresult (ng/mL)”_Diluted sample concentration (12 mg/mL).

Spikes. % CV should be=20% between triplicate wells. Record % CV. Onewell from the spike may be dropped. The remaining points must have a %difference=20%. Refer to calculation in above. Report host cellconcentration in ng/mL. This result will be used in spike recoverycalculations. The resulting concentration for the spike (ng/mL) must be±20% of the theoretical spike concentration. Record result and indicatePass or Fail. If the spike result is not within 20% of theoretical, theassay must be repeated. Mean Spike Concentration (ng/mL)×100=must be100%±20% 10 ng/mL.

Spiked Samples. % CV should be =20% between triplicate wells. Record %CV between triplicate wells. One well from each spiked sample dilutionmay be dropped. The remaining replicates must have a % difference of=20%. Refer to calculation above. Report “Spiked sample result” for eachdilution in ng/mL. Record % difference between duplicate dilutions. The% difference between dilutions should be =25%. These results will beused in the spike recovery calculations.

Calculate % Spike Recovery for each dilution set using the formulabelow: % Spike Recovery=Spiked sample value−Non-Spiked Sample Value×100Spike Value. NOTE: (1) If non-spiked sample value OD's fall below the2.5 ng/mL standard consider value as zero in % spike recoverycalculation. % Spike recovery must be 100%±50% (50%-150%) for eachdilution for each sample. Record results and Pass/Fail.

Control. % CV should be =20% between triplicate wells. Record % CVresult. One well from the control may be dropped. The remainingreplicates must have a % difference of =20%. Refer to calculation above.Report Host Cell concentration in the control in ng/mL. Calculate HostCell concentration in ng/mg as follows: Host Cell Protein(ng/mg)=Control Host Cell Protein result in ng/mL.

4. Determination of Protein A Concentration in anti-IL-12 AntibodyCompositions

In this ELISA, plates are coated with Chicken Anti-Protein A andincubated. Non-specific sites are blocked with casein in PBS. Plates arewashed in 1×PBS+0.1% Triton X-100 to remove unbound material. Samplesand Cys-rprotein A standards are diluted in 1×PBS+4.1% Triton X+10%Casein. The solutions are denatured by heating at 95° C.±2° C.,separating Protein A from ABT-874. The solutions are then added to theplate and incubated. Unbound material is washed off with 1×PBS+0.1%Triton X-100. Biotinylated Chicken Anti-Protein A is added to themicrotiter plate and incubated. The plate is washed to remove unboundmaterial and Neutravidin—Peroxidase conjugate is added.

The Neutravidin will bind to the Biotinylated Chicken Anti-Protein Athat has bound to the wells. The plate is washed again to remove theunbound Neutravidin and K-Blue (tetramethylbenzidine (TMB)) substrate isadded to the plate. The substrate is hydrolyzed by the bound Neutravidinproducing a blue color. The reaction is stopped with Phosphoric Acid,changing color to yellow. The intensity of the yellow color in the wellsis directly proportional to the concentration of Protein A present inthe wells.

Preparation of Reagents and Solutions Casein bottles must be warmed to37° C.±2° C.; sonicated for 2 minutes, and aliquoted. Aliquots are to bestored at nominal 4° C. When assay is to be run, the number of caseinaliquots needed, should be placed at 37° C.±2° C. The coating buffer andsubstrate are used cold (taken from nominal 4° C. right before use).

50 mM Sodium Bicarbonate (Coating Buffer), pH 9.4. To a 1 L beaker add:900 mL Milli-Q water 4.20 g±0.01 g Sodium Bicarbonate. Stir untilcompletely dissolved. Adjust pH to 9.4 with 1 N NaOH. Transfer to a 1 Lvolumetric flask and bring to volume with Milli-Q water. Mix byinversion until homogeneous. Filter through a 0.22 CA μm sterile filterunit. Store at nominal 4° C. for up to 7 days from the date ofpreparation.

104 M Na₂HPO₄*7H2O, 1.37 M NaCl, 0.027 M KCl, 0.0176 M KH₂PO₄,pH=6.8-6.9. (10×PBS): Add approximately 400 mL of Milli-Q water to aglass beaker. Add 13.94 g±0.01 g of Na₂HPO₄×7H2O. Add 40.0 g±0.1 g ofNaCl. Add 1.00 g±0.01 g of KCl. Add 1.20 g±0.01 g of KH₂PO₄. Stir untilhomogeneous. Transfer to a 500 mL volumetric flask. QS to 500 mL volumewith Milli-Q water. Mix by inversion. Filter through a 0.2 CA μm sterilefilter unit. Store at room temperature for up to 7 days.

1×PBS+0.1% Triton X-100, pH 7.40: (Plate Wash Buffer). In a 4 Lgraduated cylinder, mix 400 mL 10×PBS (see above) with 3500 mL Milli-QWater. Check pH, and adjust if necessary to 7.40±0.05 with 1 N HCl or 1N NaOH. Bring to volume with Milli-Q water. Tightly parafilm thecylinder and mix by inversion until homogeneous. Transfer to a 4 Lbottle. Remove 4 mL of the 1×PBS and discard. Add 4 mL of triton X-100to the 3996 mL of 1×PBS. Place on stir plate and stir to completelydissolve. Store at room temperature for up to 7 days.

Chicken Anti-Protein A Coating Antibody. Take out one aliquot ofantibody per plate at time of use. To qualify new lots of ChickenAnti-Protein A, it may be necessary to use and qualify ChickenAnti-Protein A-Biotin Conjugated (prepared from the same lot of coating)together. Immediately before use: Dilute antibody mixture in cold 50 mMSodium Bicarbonate to the concentration determined during coatingqualification. For example: If during qualification the concentration ofcoating to load on the plate was determined to be 6 μg/mL and if thestock concentration is 3000 μg/mL, then add 24 μLs coating antibody to11976 μLs cold coating buffer. Mix gently by inversion.

Biotinylated Chicken anti Protein A. Take out one aliquot of antibodyper plate at time of use. To qualify new lots of Chicken Anti-ProteinA-Biotin Conjugated, it may be necessary to use and qualify it with thesame lot of Chicken Anti-Protein A it was prepared from. Immediatelybefore use: Dilute biotinylated antibody in 37° C.±2° C. Casein to theconcentration determined during biotinylated antibody qualification. Forexample: If during qualification the concentration of biotinylatedantibody to load on the plate was determined to be 4 μg/mL and if thestock concentration is 1000 μg/mL, then add 48 μLs biotinylated antibodyto 11952 μLs 37° C.±2° C. Casein. Mix gently by inversion.

Neutravidin-HRP. Reconstitute new lots (2 mg/vial) to 1 mg/mL asfollows: Add 400 μL of Milli-Q water to the vial, then add 1600 μL1×PBS, for a total of 2 mL. Vortex gently to mix. Store at nominal −80°C. Prepare aliquots with desired volume so that 1 aliqout per plate isused. Prepare in polypropylene tube. Assign expiration date of 6 monthsfrom the date of preparation. For example, if the working concentrationwas determined to be 0.1 μg/mL then prepare as follows. Immediatelybefore use, thaw an aliquot of Neutravidin-HRP at room temperature.Dilute the 1 mg/mL Neutravidin solution to 0.01 mg/mL (10 μg/mL) with37° C.±2° C. Casein. For example: Dilute X10, add 50 μL of neutravidinto 450 μL of Casein. Vortex gently to mix, X10 again, add 100 μL of X10neutravidin to 900 μL of Casein. Vortex gently to mix. Further dilutethe 10 μg/mL solution to 0.1 μg/mL with 37° C.±2° C. Casein. Forexample: Dilute X100, add 120 μL neutravidin (10 μg/mL) to 11880 μL ofCasein. Invert several times gently to mix.

Stop Solution (Purchased 1 N Phosphoric Acid is used.) Store at ambienttemperature for up to 1 year from the date of receipt. Dilution Buffer(1×PBS+4.1% Triton X100+10% Casein, pH 7.4). Add 86 mL of 1×PBS+0.1%Triton X100, pH 7.4 (from Step 5.3) to a beaker or flask, add 4 mL ofTriton X-100, and 10 mL of Blocker Casein in PBS, and stir todissolve/mix. It may take 20 to 30 minutes to dissolve triton. Thisequals a 1×PBS+4.1% Triton X100+10% Casein, pH 7.4 solution. Filterthrough a 0.22 CA μm sterile filter unit. Prepare fresh for each use.This is enough for 1 plate.

Protein A Standards (Antigen Standards). NOTE: Stocks stored atnominal—20° C. in 70 μL aliquots. Thaw an aliquot on ice. Perform serialdilutions according to the examples in the table below polypropylenetubes using Dilution buffer (see above) using the concentration statedon the manufacturers COA: For example if COA states stock concentrationis 2.1 mg/mL (2100000 ng/mL) then: Thaw samples on ice. In polypropylenemicrocentrifuge tubes, dilute final bulk samples to 20 mg/mL in DilutionBuffer (above). Perform 2 separate dilutions. Record concentration. Usethe solutions below to prepare spiked samples and to prepare the 10mg/mL solutions. For example: Conc. (mg/mL) Vol. μL of X mg/mL solutionVol. of diluent (μL) Serial Dilution From 120 stock sample. Inpolypropylene microcentrifuge tubes, further dilute the 20 mg/mLsolutions to 10 mg/mL in Dilution Buffer.

Preparation of Spike. In a polypropylene microcentrifuge tube, prepare a0.296 ng/mL Protein A spike from the 0.593 ng/mL standard prepared abovein Step 6.1 by diluting it 2× with Dilution Buffer. Perform a singledilution. Triplicate wells for the 0.296 ng/mL spike solution will beloaded onto the plate. Use the 0.593 ng/mL standard solution from Step6.1 for spiking samples.

Preparation of Spiked Samples. In Polypropylene Microcentrifuge Tubes,Spike 500 μL of each 20 mg/mL final bulk solution with 500 μL of the0.593 ng/mL spike solution. Hold for denaturation. Triplicate wells foreach spiked sample solution will be loaded on the plate for a total of 6wells.

Preparation of Control. Obtain a lot of ABT-874 Drug Substance. Prepare150 μL aliquots and store frozen at nominal −80° C. for three years fromthe date aliquoted.

Working Control: Thaw an aliquot of control on ice. In a polypropylenemicrocentrifuge tube, dilute control to 10 mg/mL with Dilution Buffer tohave a final volume of 1000 μLs. Prepare a single dilution. Hold fordenaturation. Triplicate wells of control will be loaded onto the plate.

Denaturation. For plate blanks, add 1000 μLs of dilution buffer tomicrocentrifuge tubes equal to the number of blanks that will be run onthe plate. The caps of the tubes may be parafilmed to prevent them frompopping open during heating or a second rack may be placed on top ofthem to keep caps closed. Heat standards, non-spiked samples, spikedsamples, spike, blanks, and control, at 95° C.±2° C. for 15 minutes.Remove parafilm from tubes during cooling, if used. Allow to cool for 15minutes, and centrifuge for 5 minutes at approximately 10000 rpm.Transfer 700 μLs of the supernatant into microtubes to load on plate. Becareful not to disturb the triton/protein pellet.

Plate Washer Instructions and Waterbath Set-Up. Fill plate wash bottlewith plate wash buffer (refer to Step 5.3, 1×PBS+0.1% Triton X-100).Prime plate washer. Check the following parameters: Parameters should beset to: Plate Type: 1 For each Cycle (a total of 4 cycles): Asp speed:10 mm/s; Volume: 400 μls; Soak Time: 5 seconds; Asp. Time: 6 seconds.Turn on waterbath and set to 95° C. Allow waterbath temperature toequilibrate to 95° C.±2° C. for at least 30 minutes.

Assay Procedure: A Checklist can be used as a guide by checking offsteps as they are completed. Additionally, record all equipment usedduring the assay. The amount of Casein aliquots to be used for each daythe assay will be run must be placed at 37° C.±2° C. The coating Bufferand substrate are used cold. Prepare standard, sample, control, spike,and spiked samples prior to and during blocking incubation. It may takelonger than the 1 hour block incubation to prepare dilutions, transferto eppendorf tubes, denature for 15 minutes, cool for 15 minutes,centrifuge for 5 minutes, and to transfer to microtubes. Allow at least40 minutes prior to blocking plates. Samples, Spiked Samples, Standards,Control, Assay Spike, and Blanks, are loaded on the plate horizontallyfrom rows B through G using a 12 channel pipette. Standards are loadedfrom high to low concentration. Plate coating, biotin addition,neutravidin addition, substrate addition, and stop solution addition aredone vertically from columns 2 through 11.

Coat plates with 100 μL/well of coating antibody in cold 50 mM SodiumBicarbonate. Tap the side of the plate until the coating solution coversthe bottom of the wells uniformly, cover with sealing tape and incubateat nominal 4° C. while shaking on plate shaker (or equivalent) at speed3.

After overnight incubation, remove plate from refrigerator and allow toequilibrate to room temperature. Shake out coating. Blot plate on papertowels. Block with 300 μL/well of 37° C.±2° C. Casein, cover withsealing tape and incubate at 37° C.±2° C. while shaking on Lab-lineEnviron plate shaker (or equivalent) at 80 rpm±5 rpm for 1 hour±10minutes.

Prepare standard, sample, control, spike, and spiked samples prior toand during blocking incubation. Wash the plate 4 times with Wash Buffer.Blot plate on paper towels. Using an 8-channel pipette, pipet 100μL/well of denatured standards, samples, spikes, spiked samples, blanks,and control into triplicate wells of the plate. The outside wells of theplate are not used, add non-treated dilution buffer to these wells.Cover with sealing tape and incubate at 37° C.±2 C while shaking onLab-line Environ plate shaker (or equivalent) at 80 rpm±5 rpm for 2hours. Fill out a template to use as a guide when loading plate.

Plate Reader Set-Up. Wash the plate 4 times with Wash Buffer. Blot plateon paper towels. Add 100 μL/well biotinylated antibody. Cover withsealing tape and incubate at 37° C.±2° C. while shaking on Lab-lineEnviron plate shaker (or equivalent) at 80 rpm±5 rpm for 1 hour.

Wash the plate 4 times with Wash Buffer. Blot plate on paper towels. Add100 pt/well Neutravidin-HRP conjugate solution. Start timer as soon asneutravidin is added to the last row. Cover with sealing tape andincubate at 37° C.±2° C. while shaking on Lab-line Environ plate shaker(or equivalent) at 80 rpm±5 rpm for 30 minutes. Wash the plate 4 timeswith Wash Buffer. Blot plate on paper towels. Add 100 μL/well coldK-Blue substrate, cover with sealing tape and incubate at roomtemperature for 10 minutes (start timer as soon as substrate is added tofirst row), while shaking speed 3 on Lab-line titer plate shaker (orequivalent). Stop the reaction by adding 100 μL/well 1 N PhosphoricAcid. Place plate on a plate shaker at speed 3 for 3 minutes. Read plateat 450 nm.

Data Analysis and Calculations NOTE: Only samples, spikes, spikedsamples, and control, with optical densities falling within thepractical quantitation limit of the standard curve and meeting the % CVor % difference criteria stated below, are accepted. If sample OD's fallbelow standard curve, result should be reported as less than 0.18 ng/mL(assay LOQ). This value should then be divided by the diluted sampleconcentration (10 mg/mL) to report value in ng/mg. If the sample is highin Protein A concentration causing the non-spiked and/or the spikedsample to be above standard curve (2 ng/mL), then dilute further to bewithin the standard curve. This value should then be divided by thediluted sample concentration to report value in ng/mg. For spikerecovery calculations, subtract non-spiked sample value (ng/mL) fromspiked sample value (ng/mL) even when the non-spiked sample value(ng/mL) is below the curve. If value is negative or ‘range’ is obtainedthen consider non-spiked sample as zero for spike recovery calculations.

Standard Curve. Standard concentrations should be entered into theprotocol template. A quadratic curve fit is used. Coefficient ofdetermination must be=0.99 and the % CV between triplicate wells must be=20%. If this criteria is not met: One standard (1 level, 3 wells) maybe dropped. If the 0.18 ng/mL is dropped, only samples and spikedsamples with optical densities falling within the 0.26 ng/mL and 2 ng/mL(the remaining standard curve points) optical densities are acceptable.Additionally, for the triplicates of each standard level, if a singlewell is clearly contaminated or shows low binding, it may be dropped. Ifa well is dropped from a standard level, the remaining replicates musthave a % difference=20%. The % CV for the lowest standard, which showsOD values close to the background (blanks) of the plate, should be =30%.If one well is dropped, the % difference for the remaining replicatesmust be =35%. If the lowest standard is dropped, only samples and spikedsamples with optical densities falling within the remaining standardcurve level optical densities are acceptable.

Calculate % Difference as follows: % Difference=(Abs. (result dilution1−result dilution 2)/mean value)×100%. The assay must be repeated if thestandards do not meet the above criteria. Report % CV and/or %difference values and standard Curve Coefficient of determinationresults.

Samples. % CV should be =20% between triplicate wells. Report % CVbetween triplicate wells. One well from each sample dilution may bedropped. The remaining replicates must have a % difference of =20%.Note: If non-spiked sample OD is below lowest standard OD the %difference criteria does not apply to the non-spiked results. Refer tocalculation above.

Report “Non-spiked sample result” for each dilution in ng/mL. Thesevalues will be used in spike recovery calculations. Calculate the mean“Non-spiked sample result (ng/mL)” and the % difference betweendilutions. Report results. % Difference between dilutions must be =25%.Calculate actual Protein A Concentration in ng/mg from the mean (ng/mL)value as follows: Protein A (ng/mg)=Mean “Non-spiked sample result(ng/mL)” Diluted sample concentration (10 mg/mL). Record result.

Spikes. % CV should be =20% between triplicate wells. Record % CV. Onewell from the spike may be dropped. The remaining points must have a %difference=20%. Refer to calculation above. Report protein Aconcentration in ng/mL. This result will be used in spike recoverycalculations. The resulting concentration for the spike (ng/mL) must be±20% of the theoretical spike concentration. Record result and indicatePass or Fail. If the spike result is not within 20% of theoretical, theassay must be repeated. Mean Spike Concentration (ng/mL)×100=must be100%±20% 0.296 ng/mL

Spiked Samples. % CV should be =20% between triplicate wells. Record %CV between triplicate wells. One well from each spiked sample dilutionmay be dropped. The remaining replicates must have a % difference of=20%. Refer to calculation above. Report “Spiked sample result” for eachdilution in ng/mL. Record % difference between duplicate dilutions. The% difference between dilutions should be =25%. These results will beused in the spike recovery calculations. Calculate % Spike Recovery foreach dilution set using the formula below: % Spike Recovery=Spikedsample value−Non-Spiked Sample Value×100. Spike Value NOTE: For spikerecovery calculations, subtract non-spiked sample value (ng/mL) fromspiked sample value (ng/mL) even when the non-spiked sample value(ng/mL) is below the curve. If value is negative or ‘range’ is obtainedthen consider non-spiked sample as zero for spike recovery calculations.% Spike recovery must be 100%±50% (50%-150%) for each dilution for eachsample. Record results and Pass/Fail.

Control. % CV should be =20% between triplicate wells. Record % CVresult. One well from the control may be dropped. The remainingreplicates must have a % difference of =20%.

Various publications are cited herein, the contents of which are herebyincorporated by reference in their entireties.

1. A method for producing a host cell-protein (HCP) reduced antibodypreparation from a sample mixture comprising an antibody and at leastone HCP, said method comprising: (a) subjecting said sample matrix to areduction in pH thus forming a primary recovery sample, wherein saidreduction in pH is from about 3 to about 4; (b) adjusting said primaryrecovery sample to a pH of about 6.0 to about 8 followed by contactingsaid primary recovery sample to an affinity chromatography resin andcollecting an affinity chromatography sample; (c) contacting saidaffinity chromatography sample to an ion exchange resin and collectingan ion exchange sample; (d) contacting said ion exchange sample to ahydrophobic interactive chromatography (HIC) resin and collecting an HICsample, wherein said HIC sample comprises said HCP-reduced antibodypreparation.
 2. The method of claim 1, wherein said reduction in pH isaccomplished by admixing a suitable acid with said sample mixture, andwherein said suitable acid is selected from the group consisting ofcitric acid, acetic acid, caprylic acid, and the like.
 3. The method ofclaim 1, wherein said affinity chromatography resin is a Protein Aresin.
 4. The method of claim 3, wherein said Protein A resin isMabSelect™ resin.
 5. The method of claim 1, wherein said ion exchangeresin is either an anion exchange resin or a cation exchange resin. 6.The method of claim 5, wherein said ion exchange resin is a cationexchange resin.
 7. The method of claim 6, wherein said cation exchangeresin is selected from the group consisting of Fractogel, carboxymethyl(CM), sulfoethyl(SE), sulfopropyl(SP), phosphate(P) and sulfonate(S). 8.The method of claim 7, wherein said cation exchange resin is Fractogel™SO₃ ⁻.
 9. The method of claim 5, wherein said ion exchange resin is ananion exchange resin.
 10. The method of claim 9, wherein said anionexchange resin is selected from the group consisting of Q sepharose,diethylaminoethyl (DEAE), quaternary aminoethyl(QAE), and quaternaryamine(Q) groups.
 11. The method of claim 11, wherein said anion exchangeresin is Q-sepharose.
 12. The method of claim 1, wherein said ionexchange step comprises a first ion exchange step and a second ionexchange step.
 13. The method of claim 1, wherein said HIC isaccomplished using a column comprising one or more hydrophobic groups.14. The method of claim 13, wherein said one or more hydrophobic groupsare selected from the group consisting of alkyl aryl-groups, and acombination thereof.
 15. The method of claim 14, wherein said column isselected from the group consisting of phenyl sepharose (such as PhenylSepharose™ 6 Fast Flow column, Phenyl Sepharose™ High Performancecolumn), Octyl Sepharose™ High Performance column, Fractogel™ EMDPropyl, Fractogel™ EMD Phenyl columns, Macro-Prep™ Methyl, Macro-Prep™t-Butyl Supports, WP HI-Propyl (C₃)™ column, and Toyopearl™ ether,phenyl or butyl columns.
 16. The method of claim 15, wherein said columncomprises phenyl sepharose.
 17. The method of claim 1, furthercomprising a filtration step, wherein said HIC sample is subjected tofiltration to remove viral particles and to facilitate buffer exchange.18. The method of claim 1, wherein said HCP-reduced antibody preparationcomprises either an anti-IL-12 antibody or an antigen-binding portionthereof, or an anti-TNFα antibody or an antigen-binding portion thereof.19. The method of claim 18, wherein said HCP-reduced antibodypreparation is an anti-IL-12 antibody or an antigen-binding portionthereof.
 20. The method of claim 18, wherein said HCP-reduced antibodypreparation is an anti-TNFα antibody or an antigen-binding portionthereof.
 21. The method of claim 19, wherein said anti-IL-12 antibody orantigen-binding portion thereof is a humanized antibody, a chimericantibody, or a multivalent antibody.
 22. The method of claim 19, whereinsaid anti-TNFα antibody or antigen-binding portion thereof is ahumanized antibody, a chimeric antibody, or a multivalent antibody. 23.The method of claim 21, wherein said anti-IL-12 antibody orantigen-binding portion thereof is a humanized antibody.
 24. The methodof claim 22, wherein said anti-TNFα antibody or antigen-binding portionthereof is a humanized antibody.
 25. The method of claim 1, wherein saidpreparation is substantially free of HCPs.
 26. The method of claim 1,further comprising depth filtration.
 27. A pharmaceutical compositioncomprising an HCP-reduced antibody preparation produced by the method ofclaim 1 and a pharmaceutically acceptable carrier.
 28. Thepharmaceutical composition of claim 27, wherein said antibody is eitheran anti-IL-12 antibody or antigen-binding portion thereof, or ananti-TNFα antibody or antigen-binding portion thereof.
 29. Thepharmaceutical composition of claim 27, wherein said composition issubstantially free of HCPs.